Compositions and methods for regulating NK cell activity

ABSTRACT

The present invention relates to novel compositions and methods for regulating an immune response in a subject. More particularly, the invention relates to specific antibodies that regulate the activity of NK cells and allow a potentiation of NK cell cytotoxicity in mammalian subjects. The invention also relates to fragments and derivatives of such antibodies, as well as pharmaceutical compositions comprising the same and their uses, particularly in therapy, to increase NK cell activity or cytotoxicity in subjects.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This patent application is a continuation of co-pending InternationalPatent Application PCT/DK2004/000470 (which is published as WO2005003168and designates the U.S.) and claims the benefit of U.S. ProvisionalPatent Applications No. 60/483,984, filed Jul. 2, 2003, and 60/545,471,filed Feb. 19, 2004, each of which being hereby entirely incorporated byreference.

FIELD OF INVENTION

The present invention relates to antibodies, antibody fragments, andderivatives thereof that cross-react with two or more inhibitoryreceptors present on the cell surface of NK cells and potentiate NK cellcytotoxicity in mammalian subjects or in a biological sample. Theinvention also relates to methods of making such antibodies, fragments,variants, and derivatives; pharmaceutical compositions comprising thesame; and the use of such molecules and compositions, particularly intherapy, to increase NK cell activity or cytotoxicity in subjects.

BACKGROUND

Natural killer (NK) cells are a sub-population of lymphocytes, involvedin non-conventional immunity. NK cells can be obtained by varioustechniques known in the art, such as from blood samples, cytapheresis,collections, etc.

Characteristics and biological properties of NK cells include theexpression of surface antigens including CD16, CD56, and/or CD57; theabsence of the alpha/beta or gamma/delta TCR complex on the cellsurface; the ability to bind to and kill cells that fail to express“self” MHC/HLA antigens by the activation of specific cytolytic enzymes;the ability to kill tumor cells or other diseased cells that express aNK activating receptor-ligand; the ability to release cytokines thatstimulate or inhibit the immune response; and the ability to undergomultiple rounds of cell division and produce daughter cells with similarbiologic properties as the parent cell. Within the context of thisinvention “active” NK cells designate biologically active NK cells, moreparticularly NK cells having the capacity of lysing target cells. Forinstance, an “active” NK cell is able to kill cells that express an NKactivating receptor-ligand and fail to express “self” MHC/HLA antigens(KIR-incompatible cells).

Based on their biological properties, various therapeutic and vaccinestrategies have been proposed in the art that rely on a modulation of NKcells. However, NK cell activity is regulated by a complex mechanismthat involves both stimulating and inhibitory signals. Accordingly,effective NK cell-mediated therapy may require both a stimulation ofthese cells and a neutralization of inhibitory signals.

NK cells are negatively regulated by major histocompatibility complex(MHC) class I-specific inhibitory receptors (Kärre et al., 1986; (Ohlenet al, 1989). These specific receptors bind to polymorphic determinantsof MHC class I molecules or HLA present on other cells and inhibit NKcell lysis. In humans, certain members of a family of receptors termedkiller Ig-like receptors (KIRs) recognize groups of HLA class I alleles.

KIRs are a large family of receptors present on certain subsets oflymphocytes, including NK cells. The nomenclature for KIRs is based uponthe number of extracellular domains (KIR2D or KIR3D) and whether thecytoplasmic tail is either long (KIR2DL or KIR3DL) or short (KIR2DS orKIR3DS). Within humans, the presence or absence of a given KIR isvariable from one NK cell to another within the NK population present ina single individual. Within the human population there is also arelatively high level of polymorphism of the KIR molecules, with certainKIR molecules being present in some, but not all individuals. CertainKIR gene products cause stimulation of lymphocyte activity when bound toan appropriate ligand. The confirmed stimulatory KIRs all have a shortcytoplasmic tail with a charged transmembrane residue that associateswith an adapter molecule having an immunostimulatory motif (ITAM). OtherKIR gene products are inhibitory in nature. All confirmed inhibitoryKIRs have a long cytoplasmic tail and appear to interact with differentsubsets of HLA antigens depending upon the KIR subtype. Inhibitory KIRsdisplay in their intracytoplasmic portion one or several inhibitorymotifs that recruit phosphatases. The known inhibitory KIR receptorsinclude members of the KIR2DL and KIR3DL subfamilies. KIR receptorshaving two Ig domains (KIR2D) identify HLA-C allotypes: KIR2DL2(formerly designated p58.2) or the closely related gene product KIR2DL3recognizes an epitope shared by group 2 HLA-C allotypes (Cw1, 3, 7, and8), whereas KIR2DL1 (p58.1) recognizes an epitope shared by thereciprocal group 1 HLA-C allotypes (Cw2, 4, 5, and 6). The recognitionby KIR2DL1 is dictated by the presence of a Lys residue at position 80of HLA-C alleles. KIR2DL2 and KIR2DL3 recognition is dictated by thepresence of an Asn residue at position 80. Importantly the greatmajority of HLA-C alleles have either an Asn or a Lys residue atposition 80. One KIR with three Ig domains, KIR3DL1 (p70), recognizes anepitope shared by HLA-Bw4 alleles. Finally, a homodimer of moleculeswith three Ig domains KIR3DL2 (p140) recognizes HLA-A3 and -A11.

Although inhibitory KIRs and other class-I inhibitory receptors (Morettaet al, 1997; Valiante et al, 1997a; Lanier, 1998) may be co-expressed byNK cells, in any given individual's NK repertoire there are cells thatexpress a single KIR and thus, the corresponding NK cells are blockedonly by cells expressing a specific class I allele group.

NK cell population or clones that are KIR mismatched, i.e., populationof NK cells that express KIR that are not compatible with a HLAmolecules of a host, have been shown to be the most likely mediators ofthe graft anti-leukemia effect seen in allogeneic transplantation(Ruggeri et al., 2002). One way of reproducing this effect in a givenindividual would be to use reagents that block the KIR/HLA interaction.

Monoclonal antibodies specific for KIR2DL1 have been shown to block theinteraction of KIR2DL1 with Cw4 (or the like) alleles (Moretta et al.,1993). Monoclonal antibodies against KIR2DL2/3 have also been describedthat block the interaction of KIR2DL2/3 with HLACw3 (or the like)alleles (Moretta et al., 1993). However, the use of such reagents inclinical situations would require the development of two therapeuticmAbs to treat all patients, regardless of whether any given patient wasexpressing class 1 or class 2 HLA-C alleles. Moreover, one would have topre-determine which HLA type each patient was expressing before decidingwhich therapeutic antibody to use, thus resulting in much higher cost oftreatment.

Watzl et al., Tissue Antigens, 56, p. 240 (2000) produced cross-reactingantibodies recognizing multiple isotypes of KIRs, but those antibodiesdid not exhibit potentiation of NK cell activity. G. M. Spaggiara etal., Blood, 100, pp. 4098-4107 (2002) carried out experiments utilizingnumerous monoclonal antibodies against various KIRs. One of thoseantibodies, NKVSF1, was said to recognize a common epitope of CD158aKIR2DL1), CD158b (KIR2DL2) and p50.3 (KIR2DS4). It is not suggested thatNKVSF1 can potentiate NK cell activity and there is no suggestion thatit could be used as a therapeutic. Accordingly, practical and effectiveapproaches in the modulation of NK cell activity have not been madeavailable so far in the art and still require HLA allele-specificintervention using specific reagents.

SUMMARY OF THE INVENTION

The present invention now provides novel antibodies, compositions, andmethods that overcome current difficulties in NK cell activation andprovide additional advantageous features and benefits. In one exemplaryaspect, the invention provides a single antibody that facilitates theactivation of human NK cells in virtually all humans. More particularly,the invention provides novel specific antibodies that cross-react withvarious inhibitory KIR groups and neutralize their inhibitory signals,resulting in potentiation of NK cell cytotoxicity in NK cells expressingsuch inhibitory KIR receptors. This ability to cross-react with multipleKIR gene products allows the antibodies of the invention to beeffectively used to increase NK cell activity in most human subjects,without the burden or expense of pre-determining the HLA type of thesubject.

In a first aspect, the invention provides antibodies, antibodyfragments, and derivatives of either thereof, wherein said antibody,fragment, or derivative cross-reacts with at least two inhibitory KIRreceptors at the surface of NK cells, neutralizes the inhibitory signalsof the NK cells, and potentiates the activity of the NK cells. Morepreferably, the antibody binds a common determinant of human KIR2DLreceptors. Even more specifically, the antibody of this invention bindsat least KIR2DL1, KIR2DL2, and KIR2DL3 receptors. For the purposes ofthis invention, the term “KIR2DL2/3” refers to either or both of theKIR2DL2 and KIR2DL3 receptors. These two receptors have a very highhomology, are presumably allelic forms of the same gene, and areconsidered by the art to be interchangeable. Accordingly, KIR2DL2/3 isconsidered to be a single inhibitory KIR molecule for the purposes ofthis invention and therefore an antibody that cross-reacts with onlyKIR2DL2 and KIR2DL3 and no other inhibitory KIR receptors is not withinthe scope of this invention.

The antibody of this invention specifically inhibits binding of MHC orHLA molecules to at least two inhibitory KIR receptors and facilitatesNK cell activity. Both activities are inferred by the term “neutralizethe inhibitory activity of KIR,” as used herein. The ability of theantibodies of this invention to “facilitate NK cell activity,”“facilitate NK cell cytotoxicity,” “facilitate NK cells,” “potentiate NKcell activity, ” “potentiate NK cell cytotoxicity,” or “potentiate NKcells” in the context of this invention means that the antibody permitsNK cells expressing an inhibitory KIR receptor on their surface to becapable of lysing cells that express on their surface a correspondingligand for that particular inhibitory KIR receptor (e.g., a particularHLA antigen). In a particular aspect, the invention provides an antibodythat specifically inhibits the binding of HLA-C molecules to KIR2DL1 andKIR2DL2/3 receptors. In another particular aspect, the inventionprovides an antibody that facilitates NK cell activity in vivo.

Because at least one of KIR2DL1 or KID2DL2/3 is present in at leastabout 90% of the human population, the more preferred antibodies of thisinvention are capable of facilitating NK cell activity against most ofthe HLA-C allotype-associated cells, respectively group 1 HLA-Callotypes and group 2 HLA-C allotypes. Thus, compositions of thisinvention may be used to effectively activate or potentiate NK cells inmost human individuals, typically in about 90% of human individuals ormore. Accordingly, a single antibody composition according to theinvention may be used to treat most human subjects, and there is seldomneed to determine allelic groups or to use antibody cocktails.

The invention demonstrates, for the first time, that cross-reactive andneutralizing antibodies against inhibitory KIRs may be generated, andthat such antibodies allow effective activation of NK cells in a broadrange of human groups.

A particular object of this invention thus resides in an antibody,wherein said antibody specifically binds both KIR2DL1 and KIR2DL2/3human receptors and reverses inhibition of NK cell cytotoxicity mediatedby these KIRs. In one embodiment, the antibody competes with monoclonalantibody DF200 produced by hybridoma DF200. Optionally said antibodywhich competes with antibody DF200 is not antibody DF200 itself.

In another embodiment, the antibody competes with monoclonal antibodyNKVSF1, optionally wherein the antibody which competes with antibodyNKVSF1 is not antibody NKVSF1.

In another embodiment, the antibody competes with antibody 1-7F9.

Preferably said antibodies are chimeric antibodies, humanizedantibodies, or human antibodies.

The term “competes with” when referring to a particular monoclonalantibody (e.g. DF200, NKVSF1, 1-7F9, EB6, GL183) means that an antibodycompetes with the monoclonal antibody (e.g. DF200, NKVSF1, 1-7F9, EB6,GL183) in a binding assay using either recombinant KIR molecules orsurface expressed KIR molecules. For example, if an antibody reducesbinding of DF200 to a KIR molecule in a binding assay, the antibody“competes” with DF200. An antibody that “competes” with DF200 maycompete with DF200 for binding to the KIR2DL1 human receptor, theKIR2DL2/3 human receptor, or both KIR2DL1 and KIR2DL2/3 human receptors.

In a preferred embodiment, the invention provides an antibody that bindsboth KIR2DL1 and KIR2DL2/3 human receptors, reverses inhibition of NKcell cytotoxicity mediated by these KIRs, and competes with DF200,1-7F9, or NKVSF1 for binding to the KIR2DL1 human receptor, theKIR2DL2/3 human receptor, or both KIR2DL1 and KIR2DL2/3 human receptors.Optionally, said antibody is not NKVSF1. Optionally, said antibody is achimeric, human, or humanized antibody.

In another embodiment, the invention provides an antibody that bindsboth KIR2DL1 and KIR2DL2/3 human receptors, reverses inhibition of NKcell cytotoxicity mediated by these KIRs, and competes with EB6 forbinding to the KIR2DL1 human receptor, competes with GL183 for bindingto the KIR2DL2/3 human receptor, or competes with both EB6 for bindingto the KIR2DL1 human receptor and GL183 for binding to the KIR2DL2/3human receptor. Optionally, said antibody is not NKVSF1; optionally saidantibody is not DF200. Optionally, said antibody is a chimeric, human,or humanized antibody.

In an advantageous aspect, the invention provides an antibody thatcompetes with DF200 and recognizes, binds to, or has immunospecificityfor substantially or essentially the same, or the same, epitope or“epitopic site” on a KIR molecule as the monoclonal antibody DF200.Preferably, said KIR molecule is a KIR2DL1 human receptor or a KIR2DL2/3human receptor.

A particular object of this invention resides in an antibody, whereinsaid antibody binds a common determinant present in both KIR2DL1 andKIR2DL2/3 human receptors and reverses inhibition of NK cellcytotoxicity mediated by these KIRs. The antibody more specificallybinds substantially the same epitope on KIR as monoclonal antibody DF200produced by hybridoma DF200 or antibody NKVSF1 produced by hybridomaNKVSF1, wherein the antibody is not NKVSF1.

In a preferred embodiment, the antibody of this invention is amonoclonal antibody. The most preferred antibody of this invention ismonoclonal antibody DF200 produced by hybridoma DF200.

The hybridoma producing antibody DF200 has been deposited at the CNCMculture collection, as Identification no. “DF200”, registration no. CNCMI-3224, registered 10 Jun. 2004, Collection Nationale de Cultures deMicroorganismes, Institut Pasteur, 25, Rue du Docteur Roux, F-75724Paris Cedex 15, France. The antibody NKVSF1 is available from Serotec(Cergy Sainte-Christophe, France), Catalog ref no. MCA2243. NKVSF1 isalso referred to as pan2D mAb herein.

The invention also provides functional fragments and derivatives of theantibodies described herein, having substantially similar antigenspecificity and activity (e.g., which can cross-react with the parentantibody and which potentiate the cytotoxic activity of NK cellsexpressing inhibitory KIR receptors), including, without limitation, aFab fragment, a Fab'2 fragment, an immunoadhesin, a diabody, a CDR, anda ScFv. Furthermore, the antibodies of this invention may be humanized,human, or chimeric.

The invention also provides antibody derivatives comprising an antibodyof the invention conjugated or covalently bound to a toxin, aradionuclide, a detectable moiety (e.g., a fluor), or a solid support.

The invention also provides pharmaceutical compositions comprising anantibody as disclosed above, a fragment thereof, or a derivative ofeither thereof. Accordingly, the invention also relates to use of anantibody as disclosed herein in a method for the manufacture of amedicament. In preferred embodiments, said medicament or pharmaceuticalcomposition is for the treatment of a cancer or other proliferativedisorder, an infection, or for use in transplantation.

In another embodiment, the invention provides a composition comprisingan antibody that binds at least two different human inhibitory KIRreceptor gene products, wherein said antibody is capable of neutralizingKIR-mediated inhibition of NK cell cytotoxicity on NK cells expressingat least one of said two different human inhibitory KIR receptors,wherein said antibody is incorporated into a liposome. Optionally saidcomposition comprises an additional substance selected from a nucleicacid molecule for the delivery of genes for gene therapy; a nucleic acidmolecule for the delivery of antisense RNA, RNAi, or siRNA forsuppressing a gene in an NK cell; or a toxin or a drug for the targetedkilling of NK cells additionally incorporated into said liposome.

The invention also provides methods of regulating human NK cell activityin vitro, ex vivo, or in vivo, comprising contacting human NK cells withan effective amount of an antibody of the invention, a fragment of suchan antibody, a derivative of either thereof, or a pharmaceuticalcomposition comprising at least one of any thereof. Preferred methodscomprise administration of an effective amount of a pharmaceuticalcompositions of this invention and are directed at increasing thecytotoxic activity of human NK cells, most preferably ex vivo or invivo, in a subject having a cancer, an infectious disease, or an immunedisease.

In further aspects, the invention provides a hybridoma comprising: (a) aB cell from a mammalian host (typically a non-human mammalian host) thathas been immunized with an antigen that comprises an epitope present onan inhibitory KIR polypeptide, fused to (b) an immortalized cell (e.g.,a myeloma cell), wherein said hybridoma produces a monoclonal antibodybinds at least two different human inhibitory KIR receptors and iscapable of at least substantially neutralizing KIR-mediated inhibitionof NK cell cytotoxicity in a population of NK cells expressing said atleast two different human inhibitory KIR receptors. Optionally, saidhybridoma does not produce monoclonal antibody NKVSF1. Preferably saidantibody binds KIR2DL1 and KIR2DL2/3 receptors. Preferably said antibodybinds a common determinant present on KIR2DL1 and KIR2DL2/3. Preferablysaid hybridoma produces an antibody that inhibits the binding of a HLA-callele molecule having a Lys residue at position 80 to a human KIR2DL1receptor, and the binding of a HLA-C allele molecule having an Asnresidue at position 80 to human KIR2DL2/3 receptors. Preferably saidhybridoma produces an antibody that binds to substantially the sameepitope as monoclonal antibody DF200 produced by hybridoma DF200 oneither KIR2DL1 or KIR2DL2/3 or both KIR2DL1 and KIR2DL2/3. An example ofsuch a hybridoma is DF200.

The invention also provides methods of producing an antibody whichcross-reacts with multiple KIR2DL gene products and which neutralizesthe inhibitory activity of such KIRs, said method comprising the stepsof:

-   immunizing a non-human mammal with an immunogen comprising a KIR2DL    polypeptide;-   preparing antibodies from said immunized mammal, wherein said    antibodies bind said KIR2DL polypeptide,-   selecting antibodies of (b) that cross-react with at least two    different KIR2DL gene products, and-   (d) selecting antibodies of (c) that potentiate NK cells. In one    embodiment, said non-human mammal is a transgenic animal engineered    to express a human antibody repertoire (e.g., a non-human mammal    comprising human immunoglobulin loci and native immunoglobulin gene    deletions, such as a Xenomousem™ (Abgenix-Fremont, Calif., USA) or    non-human mammal comprising a minilocus of human Ig-encoding genes,    such as the HuMab-mouse™ (Medarex-Princeton, N.J., USA)). .    Optionally, the method further comprises selecting an antibody that    binds a primate, preferably a cynomolgus monkey, NK cell or KIR    polypeptide. Optionally, the invention further comprises a method of    evaluating an antibody, wherein an antibody produced according to    the above method is administered to a primate, preferably a    cynomolgus monkey, preferably wherein the monkey is observed for the    presence or absence of an indication of toxicity of the antibody.

The inventors also provide a method of producing an antibody that bindsat least two different human inhibitory KIR receptor gene products,wherein said antibody is capable of neutralizing KIR-mediated inhibitionof NK cell cytotoxicity on a population of NK cells expressing said atleast two different human inhibitory KIR receptor gene products, saidmethod comprising the steps of:

-   immunizing a non-human mammal with an immunogen comprising an    inhibitory KIR polypeptide;-   preparing antibodies from said immunized animal, wherein said    antibodies bind said KIR polypeptide,-   selecting antibodies of (b) that cross-react with at least two    different human inhibitory KIR receptor gene products, and-   selecting antibodies of (c) that capable of neutralizing    KIR-mediated inhibition of NK cell cytotoxicity on a population of    NK cells expressing said at least two different human inhibitory KIR    receptor gene products, wherein the order of steps (c) and (d) is    optionally reversed and any number of the steps are optionally    repeated 1 or more times. Preferably, the inhibitory KIR polypeptide    used for immunization is a KIR2DL polypeptide and the antibodies    selected in step (c) cross-react with at least KIR2DL1 and    KIR2DL2/3. Preferably said antibody recognizes a common determinant    present on at least two different KIR receptor gene products; most    preferably said KIR are KIR2DL1 and KIR2DL2/3. Optionally, said    method further comprises selecting an antibody that binds a primate,    preferably a cynomolgus monkey, NK cell or KIR polypeptide.    Optionally, the invention further comprises a method of evaluating    an antibody, wherein an antibody produced according to the above    method is administered to a primate, preferably a cynomolgus monkey,    preferably wherein the monkey is observed for the presence or    absence of an indication of toxicity of the antibody.

Optionally, in the above-described methods, the antibody selected instep c) or d) is not NKVSF1. Preferably, the antibody prepared in step(b) in the above methods is a monoclonal antibody. Preferably theantibody selected in step (c) in the above methods inhibits the bindingof a HLA-C allele molecule having a Lys residue at position 80 to ahuman KIR2DL1 receptor, and the binding of a HLA-C allele moleculehaving an Asn residue at position 80 to human KIR2DL2/3 receptors.Preferably, the antibodies selected in step (d) in the above methodscause a potentiation in NK cytotoxicity, for example any substantialpotentiation, or at least 5%, 10%, 20%, 30% or greater potentiation inNK cytotoxicity, e.g. at least about 50% potentiation of target NKcytotoxicity (e.g., at least about 60%, at least about 70%, at leastabout 80%, at least about 85%, at least about 90%, or at least about 95%(such as, for example about 65-100%) potentiation of NK cellcytotoxicity). Preferably, the antibody binds to substantially the sameepitope as monoclonal antibody DF200 on KIR2DL1 and/or KIR2DL2/3.Optionally said methods also or alternatively comprise the additionalstep of making fragments of the selected monoclonal antibodies, makingderivatives of the selected monoclonal antibodies (e.g., by conjugationwith a radionuclide, cytotoxic agent, reporter molecule, or the like),or making derivatives of antibody fragments produced from or thatcomprise sequences that correspond to the sequences of such monoclonalantibodies.

The invention further provides a method of producing an antibody thatbinds at least two different human inhibitory KIR receptor geneproducts, wherein said antibody is capable of neutralizing KIR-mediatedinhibition of NK cell cytotoxicity on a population of NK cellsexpressing said at least two different human inhibitory KIR receptorgene products, said method comprising the steps of:

-   (a) selecting, from a library or repertoire, a monoclonal antibody    or an antibody fragment that cross-reacts with at least two    different human inhibitory KIR2DL receptor gene products, and-   (b) selecting an antibody of (a) that is capable of neutralizing    KIR-mediated inhibition of NK cell cytotoxicity in a population of    NK cells expressing said at least two different human inhibitory    KIR2DL receptor gene products. Preferably the antibody binds a    common determinant present on KIR2DL1 and KIR2DL2/3. Optionally,    said antibody selected in step (b) is not NKVSF1. Preferably, the    antibody selected in step (b) inhibits the binding of a HLA-c allele    molecule having a Lys residue at position 80 to a human KIR2DL1    receptor, and the binding of a HLA-C allele molecule having an Asn    residue at position 80 to human KIR2DL2/3 receptors. Preferably, the    antibody selected in step (b) causes a potentiation in NK    cytotoxicity, for example any substantial potentiation, or at least    5%, 10%, 20%, 30% or greater potentiation in NK cytotoxicity, e.g.    at least about 50% potentiation of target NK cytotoxicity (e.g., at    least about 60%, at least about 70%, at least about 80%, at least    about 85%, at least about 90%, or at least about 95% (such as, for    example about 65-100%) potentiation of NK cell cytotoxicity).    Preferably, the antibody binds to substantially the same epitope as    monoclonal antibody DF200 on KIR2DL1 and/or KIR2DL2/3. Optionally    the method comprises the additional step of making fragments of the    selected monoclonal antibodies, making derivatives of the selected    monoclonal antibodies, or making derivatives of selected monoclonal    antibody fragments.

Additionally, the invention provides a method of producing an antibodythat binds at least two different human inhibitory KIR receptor geneproducts, wherein said antibody is capable of neutralizing KIR-mediatedinhibition of NK cell cytotoxicity in a population of NK cellsexpressing said at least two different human inhibitory KIR receptorgene products, said method comprising the steps of:

-   culturing a hybridoma of the invention under conditions permissive    for the production of said monoclonal antibody; and-   separating said monoclonal antibody from said hybridoma. Optionally    the method comprises the additional step of making fragments of the    said monoclonal antibody, making derivatives of the monoclonal    antibody, or making derivatives of such monoclonal antibody    fragments . Preferably the antibody binds a common determinant    present on KIR2DL1 and KIR2DL2/3.

Also provided by the present invention is a method of producing anantibody that binds at least two different human inhibitory KIR receptorgene products, wherein said antibody is capable of neutralizingKIR-mediated inhibition of NK cell cytotoxicity in a population of NKcells expressing said at least two different human inhibitory KIRreceptor gene products, said method comprising the steps of:

-   isolating from a hybridoma of the invention a nucleic acid encoding    said monoclonal antibody;-   optionally modifying said nucleic acid so as to obtain a modified    nucleic acid that comprises a sequence that encodes a modified or    derivatized antibody comprising an amino acid sequence that    corresponds to a functional sequence of the monoclonal antibody or    is substantially similar thereto (e.g., is at least about 65%, at    least about 75%, at least about 85%, at least about 90%, at least    about 95% (such as about 70-99%) identical to such a sequence)    selected from a humanized antibody, a chimeric antibody, a single    chain antibody, an immunoreactive fragment of an antibody, or a    fusion protien comprising such an immunoreactive fragment; inserting    said nucleic acid or modified nucleic acid (or related nucleic acid    coding for the same amino acid sequence) into an expression vector,    wherein said encoded antibody or antibody fragment is capable of    being expressed when said expression vector is present in a host    cell grown under appropriate conditions;-   transfecting a host cell with said expression vector, wherein said    host cell does not otherwise produce immunoglobulin protein;-   culturing said transfected host cell under conditions which cause    the expression of said antibody or antibody fragment; and-   isolating the antibody or antibody fragment produced by said    transfected host cell. Preferably the antibody binds a common    determinant present on KIR2DL1 and KIR2DL2/3.

In the context of this invention isolated antibodies and relatedisolated molecules include antibodies produced by synthetic means aswell as those recovered from biological media, such as antibodiesrecovered from recombinant cells.

It will be appreciated that the invention also provides a compositioncomprising an antibody that binds at least two different humaninhibitory KIR receptor gene products, wherein said antibody is capableof neutralizing KIR-mediated inhibition of NK cell cytotoxicity in NKcells expressing at least one of said two different human inhibitory KIRreceptors, said antibody being present in an amount effective todetectably potentiate NK cell cytotoxicity in a patient or in abiological sample comprising NK cells; and a pharmaceutically acceptablecarrier or excipient. Preferably the antibody binds a common determinantpresent on KIR2DL1 and KIR2DL2/3. Said composition may optionallyfurther comprise a second therapeutic agent selected from, for example,an immunomodulatory agent, a hormonal agent, a chemotherapeutic agent,an anti-angiogenic agent, an apoptotic agent, a second antibody thatbinds to and inhibits an inhibitory KIR receptor, an anti-infectiveagent, a targeting agent, or an adjunct compound. Advantageousimmunomodulatory agents may be selected from IL-1 alpha, IL-1 beta,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,IL-13, IL-15, IL-21, TGF-beta, GM-CSF, M-CSF, G-CSF, TNF-alpha,TNF-beta, LAF, TCGF, BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF, PDGF,IFN-alpha, IFN-beta, or IFN-gamma. Examples of said chemotherapeuticagents include alkylating agents, antimetabolites, cytotoxicantibiotics, adriamycin, dactinomycin, mitomycin, carminomycin,daunomycin, doxorubicin, tamoxifen, taxol, taxotere, vincristine,vinblastine, vinorelbine, etoposide (VP-16), 5-fluorouracil (5FU),cytosine arabinoside, cyclophosphamide, thiotepa, methotrexate,camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP), aminopterin,combretastatin(s), other vinca alkyloids and derivatives or prodrugsthereof. Examples of hormonal agents include leuprorelin, goserelin,triptorelin, buserelin, tamoxifen, toremifene, flutamide, nilutamide,cyproterone bicalutamid anastrozole, exemestane, letrozole, fadrozolemedroxy, chlormadinone, megestrol, other LHRH agonists, otheranti-estrogens, other anti-androgens, other aromatase inhibitors, andother progestagens. Preferably, said second antibody that binds to andinhibits an inhibitory KIR receptor is an antibody or a derivative orfragment thereof that binds to an epitope of an inhibitory KIR receptorthat differs from the epitope bound by said antibody that binds a commondeterminant present on at least two different human inhibitory KIRreceptor gene products.

The invention further provides a method of detectably potentiating NKcell activity in a patient in need thereof, comprising the step ofadministering to said patient a composition according to the invention.A patient in need of NK cell activity potentiation can be any patienthaving a disease or disorder wherein such potentiation may promote,enhance, and/or induce a therapeutic effect (or promotes, enhances,and/or induces such an effect in at least a substantial proportion ofpatients with the disease or disorder and substantially similarcharacteristics as the patient—as may determined by, e.g., clinicaltrials). A patient in need of such treatment may be suffering from,e.g., cancer, another proliferative disorder, an infectious disease oran immune disorder. Preferably said method comprises the additional stepof administering to said patient an appropriate additional therapeuticagent selected from an immunomodulatory agent, a hormonal agent, achemotherapeutic agent, an anti-angiogenic agent, an apoptotic agent, asecond antibody that binds to and inhibits an inhibitory KIR receptor,an anti-infective agent, a targeting agent or an adjunct compoundwherein said additional therapeutic agent is administered to saidpatient as a single dosage form together with said antibody, or asseparate dosage form. The dosage of the antibody (or antibodyfragment/derivative) and the dosage of the additional therapeutic agentcollectively are sufficient to detectably induce, promote, and/orenhance a therapeutic response in the patient which comprises thepotentiation of NK cell activity. Where administered separately, theantibody, fragment, or derivative and the additional therapeutic agentare desirably administered under conditions (e.g., with respect totiming, number of doses, etc.) that result in a detectable combinedtherapeutic benefit to the patient.

Further encompassed by the present invention are antibodies of theinvention which are capable of specifically binding non-human primate,preferably monkey, NK cells and/or monkey KIR receptors. Alsoencompassed are methods for evaluating the toxicity, dosage and/oractivity or efficacy of antibodies of the invention which are candidatemedicaments. In one aspect, the invention encompasses a method fordetermining a dose of an antibody that is toxic to an animal or targettissue by administering an antibody of the invention to an non-humanprimate recipient animal having NK cells, and assessing any toxic ordeleterious or adverse effects of the agent on the animal, or preferablyon a target tissue. In another aspect, the invention is a method foridentifying an antibody that is toxic to an animal or target tissue byadministering an antibody of the invention to an non-human primaterecipient animal having NK cells, and assessing any toxic or deleteriousor adverse effects of the agent on the animal, or preferably on a targettissue. In another aspect, the invention is a method for identifying anantibody that is efficacious in treatment of an infected, disease ortumor by administering an antibody of the invention to a non-humanprimate model of infection, disease or cancer, and identifying theantibody that ameliorates the infection, disease or cancer, or a symptomthereof. Preferably said antibody of the invention is an antibody which(a) cross reacts with at least two inhibitory human KIR receptors at thesurface of human NK cells, and (b) cross-reacts with NK cells or a KIRreceptor of the non-human primate.

Further encompassed by the present invention is a method of detectingthe presence of NK cells bearing an inhibitory KIR on their cell surfacein a biological sample or a living organism, said method comprising thesteps of:

-   contacting said biological sample or living organism with an    antibody of the invention, wherein said antibody is conjugated or    covalently bound to a detectable moiety; and-   detecting the presence of said antibody in said biological sample or    living organism.    -   The invention also provides a method of purifying from a sample        NK cells bearing an inhibitory KIR on their cell surface        comprising the steps of:-   contacting said sample with an antibody of the invention under    conditions that allow said NK cells bearing an inhibitory KIR on    their cell surface to bind to said antibody, wherein said antibody    is conjugated or covalently bound to a solid support (e.g., a bead,    a matrix, etc.); and-   eluting said bound NK cells from said antibody conjugated or    covalently bound to a solid support.

In a further aspect, the invention provides an antibody, antibodyfragment, or derivative of either thereof, that comprises the lightvariable region or one or more light variable region CDRs of antibodyDF200 or antibody Pan2D as illustrated in FIG. 12. In still anotheraspect, the invention provides an antibody, antibody fragment, orderivative of either thereof that comprises a sequence that is highlysimilar to all or essentially all of the light variable region sequenceof DF200 or Pan2D or one or more of the light variable region CDRs ofone or both of these antibodies.

In a further aspect, the invention provides an antibody, antibodyfragment, or derivative of either thereof, that comprises the heavyvariable region or one or more light variable region CDRs of antibodyDF200 as illustrated in FIG. 13. In still another aspect, the inventionprovides an antibody, antibody fragment, or derivative of either thereofthat comprises a sequence that is highly similar to all or essentiallyall of the heavy variable region sequence of DF200.

These and additional advantageous aspects and features of the inventionmay be further described elsewhere herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts monoclonal antibody DF200 binding to a common determinantof various human KIR2DL receptors.

FIG. 2 depicts monoclonal antibody DF200 neutralizing theKIR2DL-mediated inhibition of KIR2DL1 positive NK cell cytotoxicity onCw4 positive target cells.

FIG. 3 depicts monoclonal antibody DF200, a Fab fragment of DF200 andKIR2DL1 or KIR2DL2/3 specific conventional antibodies neutralizing theKIR2DL-mediated inhibition of KIR2DL1 positive NK cell cytotoxicity onCw4 positive target cells and the KIR2DL-mediated inhibition ofKIR2DL2/3 positive NK cell cytotoxicity on Cw3 positive target cells.

FIG. 4 depicts reconstitution of cell lysis by NK clones of HLA Cw4positive target cells in the presence of F(ab′)2 fragments of the DF200and EB6 antibodies.

FIGS. 5 and 6 depict monoclonal antibodies DF200, NKVSF1 (pan2D), humanantibodies 1-7F9, 1-4F1, 1-6F5 and 1-6F 1, and KIR2DL1 or KIR2DL2/3specific conventional antibodies neutralizing the KIR2DL-mediatedinhibition of KIR2DL1 positive NK cell cytotoxicity on Cw4 positivetarget cells (Cw4 transfected cells in FIG. 5 and EBV cells in FIG. 6).

FIG. 7 depicts an epitope map showing results of competitive bindingexperiments obtained by surface plasmon resonance (BIAcore®) analysiswith anti-KIR antibodies to KIR2DL1, where overlapping circles designateoverlap in binding to KIR2DL1. Results show that 1-7F9 is competitivewith EB6 and 1-4F1, but not with NKVSF1 and DF200, on KIR 2DL1. Antibody1-4 F1 in turn is competitive with EB6, DF200, NKVSF1, and 1-7 F9.Antibody NKVSF1 competes with DF200, 1-4F 1, and EB6, but not 1-7F9, onKIR2DL1. DF200 competes with NKVSF1, 1-4F1, and EB6, but not 1-7F9, onKIR2DL1.

FIG. 8 depicts an epitope map showing results of competitive bindingexperiments obtained by BIAcore® analysis with anti-KIR antibodies toKIR2DL3, where overlapping circles designate overlap in binding toKIR2DL3. Results show that 1-4F1 is competitive with NKVSF1, DF200,gl183, and 1-7F9 on KIR2DL3. 1-7F9 is competitive with DF200, gl183, and1-4F1, but not with NKVSF1, on KIR2DL3. NKVSF1 competes with DF200,1-4F1, and GL183, but not 1-7F9, on KIR2DL3. DF200 competes with NKVSF1,1-4F1, and 1-7F9, but not with GL183, on KIR2DL3.

FIG. 9 depicts an epitope map showing results of competitive bindingexperiments obtained by BIAcore® analysis with anti-KIR antibodies toKIR2DS1, where overlapping circles designate overlap in binding toKIR2DS1. Results show that antibody 1-4F1 is competitive with NKVSF1,DF200, and 1-7F9 on KIR2DS1. Antibody 1-7F9 is competitive with 1-4F1,but not competitive with DF200 and NKVSF1 on KIR2DS1. NKVSF1 competeswith DF200 and 1-4F1, but not with 1-7F9, on KIR2DS1. DF200 competeswith NKVSF1 and 1-4F1, but not with 1-7F9, on KIR2DS1.

FIG. 10 depicts NKVSF1 (pan2D) mAb titration demonstrating binding ofthe mAb to cynomolgus NK cells. Cynomolgus NK cells (NK bulk day 16)were incubated with different amount of Pan2D mAb followed byPE-conjugated goat F(ab′)2 fragments anti-mouse IgG (H+L) antibodies.The percentage of positive cells was determined with an isotypic control(purified mouse IgG1).Samples were done in duplicate. Mean fluorescenceintensity =MFI.

FIG. 12 provides a comparative alignment of the amino acid sequences ofthe light variable regions and light variable region CDRs of antibodiesDF200 and Pan2D mAb.

FIG. 13 provides the heavy variable region of antibody DF200.

DETAILED DESCRIPTION OF THE INVENTION

Antibodies

The present invention provides novel antibodies and fragments orderivatives thereof that bind common determinants of human inhibitoryKIR receptors, preferably a determinant present on at least twodifferent KIR2DL gene products, and cause potentiation of NK cellsexpressing at least one of those KIR receptors. The invention discloses,for the first time, that such cross-reacting and neutralizing antibodiescan be produced, which represents an unexpected result and opens anavenue towards novel and effective NK-based therapies, particularly inhuman subjects.

In a preferred embodiment, the antibody is not monoclonal antibodyNKVSF1.

Within the context of this invention a “common determinant” designates adeterminant or epitope that is shared by several gene products of thehuman inhibitory KIR receptors. Preferably, the common determinant isshared by at least two members of the KIR2DL receptor group. Morepreferably, the determinant is shared by at least KIR2DL1 and KIR2DL2/3.Certain antibodies of this invention may, in addition to recognizingmultiple gene products of KIR2DL, also recognize determinants present onother inhibitory KIRs, such as gene product of the KIR3DL receptorgroup. The determinant or epitope may represent a peptide fragment or aconformational epitope shared by said members. In a more specificembodiment, the antibody of this invention specifically binds tosubstantially the same epitope recognized by monoclonal antibody DF200.This determinant is present on both KIR2DL1 and KIR2DL2/3.

Within the context of this invention, the term antibody that “binds” acommon determinant designates an antibody that binds said determinantwith specificity and/or affinity.

The term “antibody,” as used herein, refers to polyclonal and monoclonalantibodies, as well as to fragments and derivatives of said polyclonaland monoclonal antibodies unless otherwise stated or clearlycontradicted by context. Depending on the type of constant domain in theheavy chains, full length antibodies typically are assigned to one offive major classes: IgA, IgD, IgE, IgG, and IgM. Several of these arefurther divided into subclasses or isotypes, such as IgG1, IgG2, IgG3,IgG4, and the like. The heavy-chain constant domains that correspond tothe difference classes of immunoglobulins are termed “alpha,” “delta,”“epsilon,” “gamma” and “mu,” respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known. IgG and/or IgM are the preferred classes of antibodiesemployed in this invention because they are the most common antibodiesin the physiological situation and because they are most easily made ina laboratory setting. Preferably the antibody of this invention is amonoclonal antibody. Because one of the goals of the invention is toblock the interaction of an inhibitory KIR and its corresponding HLAligand in vivo without depleting the NK cells, isotypes corresponding toFc receptors that mediate low effector function, such as IgG4, typicallyare preferred.

The antibodies of this invention may be produced by a variety oftechniques known in the art. Typically, they are produced byimmunization of a non-human animal, preferably a mouse, with animmunogen comprising an inhibitory KIR polypeptide, preferably a KIR2DLpolypeptide, more preferably a human KIR2DL polypeptide. The inhibitoryKIR polypeptide may comprise the full length sequence of a humaninhibitory KIR polypeptide, or a fragment or derivative thereof,typically an immunogenic fragment, i.e., a portion of the polypeptidecomprising an epitope exposed on the surface of the cell expressing aninhibitory KIR receptor. Such fragments typically contain at least about7 consecutive amino acids of the mature polypeptide sequence, even morepreferably at least about 10 consecutive amino acids thereof. Fragmentstypically are essentially derived from the extra-cellular domain of thereceptor. Even more preferred is a human KIR2DL polypeptide whichincludes at least one, more preferably both, extracellular Ig domains,of the full length KIRDL polypeptide and is capable of mimicking atleast one conformational epitope present in a KIR2DL receptor. In otherembodiments, said polypeptide comprises at least about 8 consecutiveamino acids of an extracellular Ig domain of amino acid positions 1-224of the KIR2DL1 polypeptide (amino acid numbering of according to PROWweb site describing the KIR gene family,http://www.ncbi.nlm.nih.gov/prow/guide/1326018082. htm)

In a most preferred embodiment, the immunogen comprises a wild-typehuman KIR2DL polypeptide in a lipid membrane, typically at the surfaceof a cell. In a specific embodiment, the immunogen comprises intact NKcells, particularly intact human NK cells, optionally treated or lysed.

The step of immunizing a non-human mammal with an antigen may be carriedout in any manner well known in the art for stimulating the productionof antibodies in a mouse (see, for example, E. Harlow and D. Lane,Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1988)). The immunogen is then suspended ordissolved in a buffer, optionally with an adjuvant, such as completeFreund's adjuvant. Methods for determining the amount of immunogen,types of buffers and amounts of adjuvant are well known to those ofskill in the art and are not limiting in any way on the presentinvention. These parameters may be different for different immunogens,but are easily elucidated.

Similarly, the location and frequency of immunization sufficient tostimulate the production of antibodies is also well known in the art. Ina typical immunization protocol, the non-human animals are injectedintraperitoneally with antigen on day I and again about a week later.This is followed by recall injections of the antigen around day 20,optionally with adjuvant such as incomplete Freund's adjuvant. Therecall injections are performed intravenously and may be repeated forseveral consecutive days. This is followed by a booster injection at day40, either intravenously or intraperitoneally, typically withoutadjuvant. This protocol results in the production of antigen-specificantibody-producing B cells after about 40 days. Other protocols may alsobe utilized as long as they result in the production of B cellsexpressing an antibody directed to the antigen used in immunization.

For polyclonal antibody preparation, serum is obtained from an immunizednon-human animal and the antibodies present therein isolated bywell-known techniques. The serum may be affinity purified using any ofthe immunogens set forth above linked to a solid support so as to obtainantibodies that react with inhibitory KIR receptors.

In an alternate embodiment, lymphocytes from an unimmunized non-humanmammal are isolated, grown in vitro, and then exposed to the immunogenin cell culture. The lymphocytes are then harvested and the fusion stepdescribed below is carried out.

For monoclonal antibodies, the next step is the isolation of splenocytesfrom the immunized non-human mammal and the subsequent fusion of thosesplenocytes with an immortalized cell in order to form anantibody-producing hybridoma. The isolation of splenocytes from anon-human mammal is well-known in the art and typically involvesremoving the spleen from an anesthetized non-human mammal, cutting itinto small pieces and squeezing the splenocytes from the splenic capsuleand through a nylon mesh of a cell strainer into an appropriate bufferso as to produce a single cell suspension. The cells are washed,centrifuged and resuspended in a buffer that lyses any red blood cells.The solution is again centrifuged and remaining lymphocytes in thepellet are finally resuspended in fresh buffer.

Once isolated and present in single cell suspension, the lymphocytes canbe fused to an immortal cell line. This is typically a mouse myelomacell line, although many other immortal cell lines useful for creatinghybridomas are known in the art. Preferred murine myeloma lines include,but are not limited to, those derived from MOPC-21 and MPC-11 mousetumors available from the Salk Institute Cell Distribution Center, SanDiego, Calif. U.S.A., X63 Ag8653 and SP-2 cells available from theAmerican Type Culture Collection, Rockville, Md. U.S.A. The fusion iseffected using polyethylene glycol or the like. The resulting hybridomasare then grown in selective media that contains one or more substancesthat inhibit the growth or survival of the unfused, parental myelomacells. For example, if the parental myeloma cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (HAT medium), which substances prevent thegrowth of HGPRT-deficient cells.

Hybridomas are typically grown on a feeder layer of macrophages. Themacrophages are preferably from littermates of the non-human mammal usedto isolate splenocytes and are typically primed with incomplete Freund'sadjuvant or the like several days before plating the hybridomas. Fusionmethods are described in Goding, “Monoclonal Antibodies: Principles andPractice,” pp. 59-103 (Academic Press, 1986), the disclosure of which isherein incorporated by reference.

The cells are allowed to grow in the selection media for sufficient timefor colony formation and antibody production. This is usually betweenabout 7 and about 14 days. The hybridoma colonies are then assayed forthe production of antibodies that cross-react with multiple inhibitoryKIR receptor gene products. The assay is typically a colorimetricELISA-type assay, although any assay may be employed that can be adaptedto the wells that the hybridomas are grown in. Other assays includeimmunoprecipitation and radioimmunoassay. The wells positive for thedesired antibody production are examined to determine if one or moredistinct colonies are present. If more than one colony is present, thecells may be re-cloned and grown to ensure that only a single cell hasgiven rise to the colony producing the desired antibody. Positive wellswith a single apparent colony are typically re-cloned and re-assayed toinsure only one monoclonal antibody is being detected and produced.Antibodies may also be produced by selection of combinatorial librariesof immunoglobulins, as disclosed for instance in Ward et al., Nature,341 (1989) p. 544).

The antibodies of this invention are able to neutralize the KIR-mediatedinhibition of NK cell cytotoxicity; particularly inhibition mediated byKIR2DL receptors and more particularly at least both the KIR2DL1 andKIR2DL2/3 inhibition. These antibodies are thus “neutralizing” or“inhibitory” antibodies, in the sense that they block, at leastpartially and detectably, the inhibitory signaling pathway mediated byKIR receptors when they interact with MHC class I molecules. Moreimportantly, this inhibitory activity is displayed with respect toseveral types of inhibitory KIR receptors, preferably several KIR2DLreceptor gene products, and more preferably at least both KIR2DL1 andKIR2DL2/3 so that these antibodies may be used in various subjects withhigh efficacy. Inhibition of KIR-mediated inhibition of NK cellcytotoxicity can be assessed by various assays or tests, such as bindingor cellular assays.

Once an antibody that cross-reacts with multiple inhibitor KIR receptorsis identified, it can be tested for its ability to neutralize theinhibitory effect of those KIR receptors in intact NK cells. In aspecific variant, the neutralizing activity can be illustrated by thecapacity of said antibody to reconstitute lysis by KIR2DL-positive NKclones of HLA-C positive targets. In another specific embodiment, theneutralizing activity of the antibody is defined by the ability of theantibody to inhibit the binding of HLA-C molecules to KIR2DL1 andKIR2DL3 (or the closely related KIR2DL2) receptors, further preferablyas it is the capacity of the antibody to alter:

-   the binding of a HLA-C molecule selected from Cw1, Cw3, Cw7, and Cw8    (or of a HLA-C molecule having an Asn residue at position 80) to    KIR2DL2/3; and-   the binding of a HLA-C molecule selected from Cw2, Cw4, Cw5 and Cw6    (or of a HLA-C molecule having a Lys residue at position 80) to    KIR2DL1.

In another variant, the inhibitory activity of an antibody of thisinvention can be assessed in a cell based cytotoxicity assay, asdisclosed in the Examples provided herein.

In another variant, the inhibitory activity of an antibody of thisinvention can be assessed in a cytokine-release assay, wherein NK cellsare incubated with the test antibody and a target cell line expressingone HLA-C allele recognized by a KIR molecule of the NK population, tostimulate NK cell cytokine production (for example IFN-γ and/or GM-CSFproduction). In an exemplary protocol, IFN-γ production from PBMC isassessed by cell surface and intracytoplasmic staining and analysis byflow cytometry after about 4 days in culture. Briefly, Brefeldin A(Sigma Aldrich) can be added at a final concentration of about 5 μg/mlfor the least about 4 hours of culture. The cells can then incubatedwith anti-CD3 and anti-CD56 mAb prior to permeabilization (IntraPrep™;Beckman Coulter) and staining with PE-anti-IFN-γ or PE-IgG1(Pharmingen). GM-CSF and IFN-γ production from polyclonal activated NKcells can be measured in supernatants using ELISA (GM-CSF: DuoSet Elisa,R&D Systems, Minneapolis, MN; IFN-γ: OptE1A set, Pharmingen).

Antibodies of this invention may partially or fully neutralize theKIR-mediated inhibition of NK cell cytotoxicity. The term “neutralizeKIR-mediated inhibition of NK cell cytotoxicity,” as used herein meansthe ability to increase to at least about 20%, preferably to at leastabout 30%, at least about 40%, at least about 50% or more (e.g., about25-100%) of specific lysis obtained at the same ratio with NK cells orNK cell lines that are not blocked by their KIR, as measured by aclassical chromium release test of cytotoxicity, compared with the levelof specific lysis obtained without antibody when an NK cell populationexpressing a given KIR is put in contact with a target cell expressingthe cognate MHC class I molecule (recognized by the KIR expressed on NKcell). For example, preferred antibodies of this invention are able toinduce the lysis of matched or HLA compatible or autologous target cellpopulations, i.e., cell populations that would not be effectively lysedby NK cells in the absence of said antibody. Accordingly, the antibodiesof this invention may also be defined as facilitating NK cell activityin vivo.

Alternatively, the term “neutralize KIR mediated inhibition” means thatin a chromium assay using an NK cell clone or transfectant expressingone or several inhibitory KIRs and a target cell expressing only one HLAallele that is recognized by one of the KIRs on the NK cell, the levelof cytotoxicity obtained with the antibody should be at least about 20%,preferably at least about 30%, at least about 40%, at least about 50%(e.g., about 25-100%), or more of the cytotoxicity obtained with a knownblocking anti MHC class I molecule, such as W6/32 anti MHC class Iantibody.

In a specific embodiment, the antibody binds substantially the sameepitope as monoclonal antibody DF200 (produced by hybridoma DF200). Suchantibodies are referred to herein as “DF200 like antibodies.” In afurther preferred embodiment, the antibody is a monoclonal antibody.More preferred “DF200 like antibodies” of this invention are antibodiesother than the monoclonal antibody NKVSF1. Most preferred is monoclonalantibody DF200 (produced by hybridoma DF200).

The term “binds to substantially the same epitope or determinant as” anantibody of interest means that an antibody “competes” with saidantibody of interest. The term “binds to substantially the same epitopeor determinant as” the monoclonal antibody DF200 means that an antibody“competes” with DF200. Generally, an antibody that “binds tosubstantially the same epitope or determinant as” the monoclonalantibody of interest (e.g. DF200, NKVSF1, 17F9) means that the antibody“competes” with said antibody of interest for any one of more KIRmolecules, preferably a KIR molecule selected from the group consistingof KIR2DL1 and KIR2DL2/3. In other examples, an antibody that binds tosubstantially the same epitope or determinant on a KIR2DL1 molecule asthe antibody of interest “competes” with the antibody of interest forbinding to KIR2DL1. An antibody that binds to substantially the sameepitope or determinant on a KIR2DL2/3 molecule as the antibody ofinterest “competes” with antibody of interest for binding to KIR2DL2/3.

The term “binds to essentially the same epitope or determinant as” anantibody of interest means that an antibody “competes” with saidantibody of interest for any and all KIR molecules to which saidantibody of interest specifically binds. The term “binds to essentiallythe same epitope or determinant as” the monoclonal antibody DF200 meansthat an antibody “competes” with DF200 for any and all KIR molecules towhich DF200 specifically binds. For example, an antibody that binds toessentially the same epitope or determinant as the monoclonal antibodiesDF200 or NKVSF1 “competes” with said DF200 or NKVSF1 respectively forbinding to KIR2DL1, KIR2DL2/3, KIR2DS1 and KIR2DS2.

The identification of one or more antibodies that bind(s) tosubstantially or essentially the same epitope as the monoclonalantibodies described herein can be readily determined using any one ofvariety of immunological screening assays in which antibody competitioncan be assessed. A number of such assays are routinely practiced andwell known in the art (see, e.g., U.S. Pat. No. 5,660,827, issued Aug.26, 1997, which is specifically incorporated herein by reference). Itwill be understood that actually determining the epitope to which anantibody described herein binds is not in any way required to identifyan antibody that binds to the same or substantially the same epitope asthe monoclonal antibody described herein.

For example, where the test antibodies to be examined are obtained fromdifferent source animals, or are even of a different Ig isotype, asimple competition assay may be employed in which the control (DF200,for example) and test antibodies are admixed (or pre-adsorbed) andapplied to a sample containing both KIR2DL1 and KIR2DL2/3, each of whichis known to be bound by DF200. Protocols based upon ELISAs,radioimmunoassays, Western blotting, and the use of BIACORE analysis (asset forth, for example, in the Examples section) are suitable for use insuch simple competition studies.

In certain embodiments, one would pre-mix the control antibodies (DF200,for example) with varying amounts of the test antibodies (e.g., about1:10 or about 1:100) for a period of time prior to applying to theinhibitory KIR antigen sample. In other embodiments, the control andvarying amounts of test antibodies can simply be admixed during exposureto the KIR antigen sample. As long as one can distinguish bound fromfree antibodies (e.g., by using separation or washing techniques toeliminate unbound antibodies) and DF200 from the test antibodies (e.g.,by using species-specific or isotype-specific secondary antibodies or byspecifically labeling DF200 with a detectable label) one will be able todetermine if the test antibodies reduce the binding of DF200 to the twodifferent KIR2DL antigens, indicating that the test antibody recognizessubstantially the same epitope as DF200. The binding of the (labeled)control antibodies in the absence of a completely irrelevant antibodycan serve as the control high value. The control low value can beobtained by incubating the labeled (DF200) antibodies with unlabelledantibodies of exactly the same type (DF200), where competition wouldoccur and reduce binding of the labeled antibodies. In a test assay, asignificant reduction in labeled antibody reactivity in the presence ofa test antibody is indicative of a test antibody that recognizessubstantially the same epitope, i.e., one that “cross-reacts” with thelabeled (DF200) antibody. Any test antibody that reduces the binding ofDF200 to each of KIR2DL1 and KIR2DL2/3 antigens by at least about 50%,such as at least about 60%, or more preferably at least about 70% (e.g.,about 65-100%), at any ratio of DF200: test antibody between about 1:10and about 1:100 is considered to be an antibody that binds tosubstantially the same epitope or determinant as DF200. Preferably, suchtest antibody will reduce the binding of DF200 to each of the KIR2DLantigens by at least about 90% (e.g., about 95%).

Competition can be assessed by, for example, a flow cytometry test. Insuch a test, cells bearing a given KIR can be incubated first withDF200, for example, and then with the test antibody labeled with afluorochrome or biotin. The antibody is said to compete with DF200 ifthe binding obtained upon preincubation with saturating amount of DF200is about 80%, preferably about 50%, about 40% or less (e.g., about 30%)of the binding (as measured by mean of fluorescence) obtained by theantibody without preincubation with DF200. Alternatively, an antibody issaid to compete with DF200 if the binding obtained with a labeled DF200(by a fluorochrome or biotin) on cells preincubated with saturatingamount of test antibody is about 80%, preferably about 50%, about 40%,or less (e.g., about 30%) of the binding obtained without preincubationwith the antibody.

A simple competition assay in which a test antibody is pre-adsorbed andapplied at saturating concentration to a surface onto which both KIR2DL1and KIR2DL2/3 are immobilized also may be advantageously employed. Thesurface in the simple competition assay is preferably a BIACORE chip (orother media suitable for surface plasmon resonance analysis). Thecontrol antibody (e.g., DF200) is then brought into contact with thesurface at KIR2DL1 and KIR2DL2/3-saturating concentration and theKIR2DL1 and KIR2DL2/3 surface binding of the control antibody ismeasured. This binding of the control antibody is compared with thebinding of the control antibody to the KIR2DL1 and KIR2DL2/3-containingsurface in the absence of test antibody. In a test assay, a significantreduction in binding of the KIR2DL1 and KIR2DL2/3-containing surface bythe control antibody in the presence of a test antibody indicates thatthe test antibody recognizes substantially the same epitope as thecontrol antibody such that the test antibody “cross-reacts” with thecontrol antibody. Any test antibody that reduces the binding of control(such as DF200) antibody to each of KIR2DL1 and KIR2DL2/3 antigens by atleast about 30% or more preferably about 40% can be considered to be anantibody that binds to substantially the same epitope or determinant asa control (e.g., DF200). Preferably, such test antibody will reduce thebinding of the control antibody (e.g., DF200) to each of the KIR2DLantigens by at least about 50% (e.g., at least about 60%, at least about70%, or more). It will be appreciated that the order of control and testantibodies can be reversed: that is the control antibody can be firstbound to the surface and the test antibody is brought into contact withthe surface thereafter in a competition assay. Preferably, the antibodyhaving higher affinity for KIR2DL1 and KIR2DL2/3 antigens is bound tothe KIR2DL1 and KIR2DL2/3-containing surface first, as it will beexpected that the decrease in binding seen for the second antibody(assuming the antibodies are cross-reacting) will be of greatermagnitude. Further examples of such assays are provided in the Examplesand in, e.g., Saunal and Regenmortel, (1995) J. Immunol. Methods 183:33-41, the disclosure of which is incorporated herein by reference.

While described in the context of DF200 for the purposes ofexemplification, it will be appreciated that the above-describedimmunological screening assays can also be used to identify antibodiesthat compete with NKVSF1, 1-7F9, EB6, GL183, and other antibodiesaccording to the invention.

Upon immunization and production of antibodies in a vertebrate or cell,particular selection steps may be performed to isolate antibodies asclaimed. In this regard, in a specific embodiment, the invention alsorelates to methods of producing such antibodies, comprising:

-   immunizing a non-human mammal with an immunogen comprising an    inhibitory KIR polypeptide;-   preparing antibodies from said immunized animal, wherein said    antibodies bind said KIR polypeptide,-   selecting antibodies of (b) that cross-react with at least two    different inhibitory KIR gene products, and-   selecting antibodies of (c) that are capable of neutralizing    KIR-mediated inhibition of NK cell cytotoxicity on a population of    NK cells expressing said at least two different human inhibitory KIR    receptor gene products.    The selection of an antibody that cross-reacts with at least two    different inhibitory KIR gene products may be achieved by screening    the antibody against two or more different inhibitory KIR antigens,    for example as described above.

In a more preferred embodiment, the antibodies prepared in step (b) aremonoclonal antibodies. Thus, the term “preparing antibodies from saidimmunized animal,” as used herein, includes obtaining B-cells from animmunized animal and using those B cells to produce a hybridoma thatexpresses antibodies, as well as obtaining antibodies directly from theserum of an immunized animal. In another preferred embodiment, theantibodies selected in step (c) are those that cross-react with at leastKIR2DL1 and KIR2DL2/3.

In yet another preferred embodiment, the antibodies selected in step (d)cause at least about 10% specific lysis mediated by NK cells displayingat least one KIR recognized by the antibody, and preferably at leastabout 40% specific lysis, at least about 50% specific lysis, or morepreferably at least about 70% specific lysis (e.g., about 60-100%specific lysis), as measured in a standard chromium release assay,towards a target cell expressing cognate HLA class I molecule, comparedwith the lysis or cytotoxicity obtained at the same effector/targetratio with NK cells that are not blocked by their KIR. Alternatively,the antibodies selected in step (d) when used in a chromium assayemploying an NK cell clone expressing one or several inhibitory KIRs anda target cell expressing only one HLA allele that is recognized by oneof the KIRs on the NK clone, the level of cytotoxicity obtained with theantibody should be at least about 20% preferably at least about 30%, ormore of the cytotoxicity obtained with a blocking anti MHC class I mAbsuch as W6/32 anti MHC class I antibody.

The order of steps (c) and (d) of the immediately above-described methodcan be changed. Optionally, the method also or alternatively may furthercomprise additional steps of making fragments of the monoclonal antibodyor derivatives of the monoclonal antibody or such fragments, e.g., asdescribed elsewhere herein.

In a preferred embodiment, the non-human animal used to produceantibodies according to applicable methods of the invention is a mammal,such as a rodent (e.g., mouse, rat, etc.), bovine, porcine, horse,rabbit, goat, sheep, etc. Also, the non-human mammal may be geneticallymodified or engineered to produce “human” antibodies, such as theXenomouse™ (Abgenix) or HuMAb-Mouse™ (Medarex).

In another variant, the invention provides a method for obtaining anantibody that comprises:

-   selecting, from a library or repertoire, a monoclonal antibody, a    fragment of a monoclonal antibody, or a derivative of either thereof    that cross-reacts with at least two different human inhibitory    KIR2DL receptor gene products, and-   selecting an antibody, fragment, or derivative of (a) that is    capable of neutralizing KIR-mediated inhibition of NK cell    cytotoxicity on a population of NK cells expressing said at least    two different human inhibitory KIR2DL receptor gene products.

The repertoire may be any (recombinant) repertoire of antibodies orfragments thereof, optionally displayed by any suitable structure (e.g.,phage, bacteria, synthetic complex, etc.). Selection of inhibitoryantibodies may be performed as disclosed above and further illustratedin the examples.

According to another embodiment, the invention provides a hybridomacomprising a B cell from a non-human host, wherein said B cell producesan antibody that binds a determinant present on at least two differenthuman inhibitory KIR receptor gene products and said antibody is capableof neutralizing the inhibitory activity of said receptors. Morepreferably, the hybridoma of this aspect of the invention is not ahybridoma that produces the monoclonal antibody NKVSF1. The hybridomaaccording to this aspect of the invention can be created as describedabove by the fusion of splenocytes from the immunized non-human mammalwith an immortal cell line. Hybridomas produced by this fusion can bescreened for the presence of such a cross-reacting antibody as describedelsewhere herein. Preferably, the hybridoma produces an antibody therecognizes a determinant present on at least two different KIR2DL geneproducts, and cause potentiation of NK cells expressing at least one ofthose KIR receptors. Even more preferably, the hybridoma produces anantibody that binds to substantially the same epitope or determinant asDF200 and which potentiates NK cell activity. Most preferably, thathybridoma is hybridoma DF200 which produces monoclonal antibody DF200.

Hybridomas that are confirmed to produce a monoclonal antibody of thisinvention can be grown up in larger amounts in an appropriate medium,such as DMEM or RPMI-1640. Alternatively, the hybridoma cells can begrown in vivo as ascites tumors in an animal.

After sufficient growth to produce the desired monoclonal antibody, thegrowth media containing monoclonal antibody (or the ascites fluid) isseparated away from the cells and the monoclonal antibody presenttherein is purified. Purification is typically achieved by gelelectrophoresis, dialysis, chromatography using protein A or proteinG-Sepharose, or an anti-mouse Ig linked to a solid support such asagarose or Sepharose beads (all described, for example, in the AntibodyPurification Handbook, Amersham Biosciences, publication No.18-1037-46,Edition AC, the disclosure of which is hereby incorporated byreference). The bound antibody is typically eluted from proteinA/protein G columns by using low pH buffers (glycine or acetate buffersof pH 3.0 or less) with immediate neutralization of antibody-containingfractions. These fractions are pooled, dialyzed, and concentrated asneeded.

According to an alternate embodiment, the DNA encoding an antibody thatbinds a determinant present on at least two different human inhibitoryKIR receptor gene products, is isolated from the hybridoma of thisinvention and placed in an appropriate expression vector fortransfection into an appropriate host. The host is then used for therecombinant production of the antibody, or variants thereof, such as ahumanized version of that monoclonal antibody, active fragments of theantibody, or chimeric antibodies comprising the antigen recognitionportion of the antibody. Preferably, the DNA used in this embodimentencodes an antibody that recognizes a determinant present on at leasttwo different KIR2DL gene products, and cause potentiation of NK cellsexpressing at least one of those KIR receptors. Even more preferably,the DNA encodes an antibody that binds to substantially the same epitopeor determinant as DF200 and which potentiates NK cell activity. Mostpreferably, that DNA encodes monoclonal antibody DF200.

DNA encoding the monoclonal antibodies of the invention is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). Onceisolated, the DNA can be placed into expression vectors, which are thentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis ofmonoclonal antibodies in the recombinant host cells. Recombinantexpression in bacteria of DNA encoding the antibody is well known in theart (see, for example, Skerra et al., Curr. Opinion in Immunol., 5, pp.256 (1993); and Pluckthun, Immunol. Revs., 130, pp. 151 (1992).

Fragments and Derivatives of a Monoclonal Antibody

Fragments and derivatives of antibodies of this invention (which areencompassed by the term “antibody” or “antibodies” as used in thisapplication, unless otherwise stated or clearly contradicted bycontext), preferably a DF-200-like antibody, can be produced bytechniques that are known in the art. “Immunoreactive fragments”comprise a portion of the intact antibody, generally the antigen bindingsite or variable region. Examples of antibody fragments include Fab,Fab′, Fab′-SH, F(ab′)₂, and Fv fragments; diabodies; any antibodyfragment that is a polypeptide having a primary structure consisting ofone uninterrupted sequence of contiguous amino acid residues (referredto herein as a “single-chain antibody fragment” or “single chainpolypeptide”), including without limitation (1) single-chain Fv (scFv)molecules (2) single chain polypeptides containing only one light chainvariable domain, or a fragment thereof that contains the three CDRs ofthe light chain variable domain, without an associated heavy chainmoiety and (3) single chain polypeptides containing only one heavy chainvariable region, or a fragment thereof containing the three CDRs of theheavy chain variable region, without an associated light chain moiety;and multispecific antibodies formed from antibody fragments. Forinstance, Fab or F(ab′)2 fragments may be produced by protease digestionof the isolated antibodies, according to conventional techniques. Itwill be appreciated that immunoreactive fragments can be modified usingknown methods, for example to slow clearance in vivo and obtain a moredesirable pharmacokinetic profile the fragment may be modified withpolyethylene glycol (PEG). Methods for coupling and site-specificallyconjugating PEG to a Fab′ fragment are described in, for example, Leonget al, Cytokine 16(3):106-119 (2001) and Delgado et al, Br. J. Cancer73(2):175-182 (1996), the disclosures of which are incorporated hereinby reference.

In a particular aspect, the invention provides antibodies, antibodyfragments, and antibody derivatives comprising the light chain variableregion sequence of DF-200 as set forth in FIG. 12. In another particularaspect, the invention provides antibodies, antibody fragments, andantibody derivatives that comprise the light chain variable regionsequence of Pan2D as set forth in FIG. 12. In another aspect, theinvention provides antibodies, antibody fragments, and derivativesthereof that comprise one or more of the light variable region CDRs ofDF-200 as set forth in FIG. 12. In yet another aspect, the inventionprovides antibodies, antibody fragments, and derivatives thereof thatcomprise one or more light variable region CDRs of Pan2D as set forth inFIG. 12. Functional variants/analogs of such sequences can be generatedby making suitable substitutions, additions, and/or deletions in thesedisclosed amino acid sequences using standard techniques, which may beaided by the comparison of the sequences. Thus, for example, CDRresidues that are conserved between Pan2D and DF-200 may be suitabletargets for modification inasmuch as such residues may not contribute tothe different profiles in competition these antibodies have with respectto other antibodies disclosed herein (although Pan2D and DF-200 docompete) and thus may not contribute to the specificity of theseantibodies for their particular respective epitopes. In another aspect,positions where a residue is present in a sequence of one of theseantibodies, but not another, may be suitable for deletions,substitutions, and/or insertions.

In a particular aspect, the invention provides antibodies, antibodyfragments, and antibody derivatives comprising the heavy chain variableregion sequence of DF-200 as set forth in FIG. 13. In another aspect,the invention provides antibodies, antibody fragments, and derivativesthereof that comprise one or more of the heavy variable region CDRs ofDF-200 as set forth in FIG. 13. Functional variants/analogs of suchsequences can be generated by making suitable substitutions, additions,and/or deletions in these disclosed amino acid sequences using standardtechniques, which may be aided by the comparison of the sequences. Inanother aspect, positions where a residue is present in a sequence ofone of these antibodies, but not another, may be suitable for deletions,substitutions, and/or insertions.

Alternatively, the DNA of a hybridoma producing an antibody of thisinvention, preferably a DF-200-like antibody, may be modified so as toencode for a fragment of this invention. The modified DNA is theninserted into an expression vector and used to transform or transfect anappropriate cell, which then expresses the desired fragment.

In an alternate embodiment, the DNA of a hybridoma producing an antibodyof this invention, preferably a DF-200-like antibody, can be modifiedprior to insertion into an expression vector, for example, bysubstituting the coding sequence for human heavy- and light-chainconstant domains in place of the homologous non-human sequences (e.g.,Morrison et al., Proc. Natl. Acad. Sci. U.S.A., 81, pp. 6851 (1984)), orby covalently joining to the immunoglobulin coding sequence all or partof the coding sequence for a non-immunoglobulin polypeptide. In thatmanner, “chimeric” or “hybrid” antibodies are prepared that have thebinding specificity of the original antibody. Typically, suchnon-immunoglobulin polypeptides are substituted for the constant domainsof an antibody of the invention.

Thus, according to another embodiment, the antibody of this invention,preferably a DF-200-like antibody, is humanized. “Humanized” forms ofantibodies according to this invention are specific chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′) ₂, or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from the murineimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementary-determining region (CDR) of the recipient are replaced byresidues from a CDR of the original antibody (donor antibody) whilemaintaining the desired specificity, affinity, and capacity of theoriginal antibody. In some instances, Fv framework residues of the humanimmunoglobulin may be replaced by corresponding non-human residues.Furthermore, humanized antibodies can comprise residues that are notfound in either the recipient antibody or in the imported CDR orframework sequences. These modifications are made to further refine andoptimize antibody performance. In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDR regions correspondto those of the original antibody and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details see Jones et al., Nature, 321, pp.522 (1986); Reichmann et al., Nature, 332, pp. 323 (1988); and Presta,Curr. Op. Struct. Biol., 2, pp. 593 (1992).

Methods for humanizing the antibodies of this invention are well knownin the art. Generally, a humanized antibody according to the presentinvention has one or more amino acid residues introduced into it fromthe original antibody. These murine or other non-human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.,Nature, 321, pp. 522 (1986); Riechmann et al., Nature, 332, pp. 323(1988); Verhoeyen et al., Science, 239, pp. 1534 (1988)). Accordingly,such “humanized” antibodies are chimeric antibodies (Cabilly et al.,U.S. Pat. No. 4,816,567), wherein substantially less than an intacthuman variable domain has been substituted by the corresponding sequencefrom the original antibody. In practice, humanized antibodies accordingto this invention are typically human antibodies in which some CDRresidues and possibly some FR residues are substituted by residues fromanalogous sites in the original antibody.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of an antibody of this invention is screenedagainst the entire library of known human variable-domain sequences. Thehuman sequence which is closest to that of the mouse is then accepted asthe human framework (FR) for the humanized antibody (Sims et al., J.Immunol., 151, pp. 2296 (1993); Chothia and Lesk, J. Mol. Biol., 196,pp. 901 (1987)). Another method uses a particular framework from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework can be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.U.S.A., 89, pp. 4285 (1992); Presta et al., J. Immunol., 51, pp. 1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for multiple inhibitory KIR receptors and other favorablebiological properties. To achieve this goal, according to a preferredmethod, humanized antibodies are prepared by a process of analysis ofthe parental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

Another method of making “humanized” monoclonal antibodies is to use aXenoMouse(® (Abgenix, Fremont, Calif.) as the mouse used forimmunization. A XenoMouse is a murine host according to this inventionthat has had its immunoglobulin genes replaced by functional humanimmunoglobulin genes. Thus, antibodies produced by this mouse or inhybridomas made from the B cells of this mouse, are already humanized.The XenoMouse is described in U.S. Pat. No. 6,162,963, which is hereinincorporated in its entirety by reference. An analogous method can beachieved using a HuMAb-Mouse® (Medarex).

Human antibodies may also be produced according to various othertechniques, such as by using, for immunization, other transgenic animalsthat have been engineered to express a human antibody repertoire(Jakobovitz et al., Nature 362 (1993) 255), or by selection of antibodyrepertoires using phage display methods. Such techniques are known tothe skilled person and can be implemented starting from monoclonalantibodies as disclosed in the present application.

The antibodies of the present invention, preferably a DF-200-likeantibody, may also be derivatized to “chimeric” antibodies(immunoglobulins) in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in the originalantibody, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (Cabilly et al., supra; Morrison et al., Proc. Natl.Acad. Sci. U.S.A., 81, pp. 6851 (1984)).

Other derivatives within the scope of this invention includefunctionalized antibodies, i.e., antibodies that are conjugated orcovalently bound to a toxin, such as ricin, diphtheria toxin, abrin andPseudomonas exotoxin; to a detectable moiety, such as a fluorescentmoiety, a radioisotope or an imaging agent; or to a solid support, suchas agarose beads or the like. Methods for conjugation or covalentbonding of these other agents to antibodies are well known in the art.

Conjugation to a toxin is useful for targeted killing of NK cellsdisplaying one of the cross-reacting KIR receptors on its cell surface.Once the antibody of the invention binds to the cell surface of suchcells, it is internalized and the toxin is released inside of the cell,selectively killing that cell. Such use is an alternate embodiment ofthe present invention.

Conjugation to a detectable moiety is useful when the antibody of thisinvention is used for diagnostic purposes. Such purposes include, butare not limited to, assaying biological samples for the presence of theNK cells bearing the cross-reacting KIR on their cell surface anddetecting the presence of NK cells bearing the cross-reacting KIR in aliving organism. Such assay and detection methods are also alternateembodiments of the present invention.

Conjugation of an antibody of this invention to a solid support isuseful as a tool for affinity purification of NK cells bearing thecross-reacting KIR on their cell surface from a source, such as abiological fluid. This method of purification is another alternateembodiment of the present invention, as is the resulting purifiedpopulation of NK cells.

In an alternate embodiment, an antibody that binds a common determinantpresent on at least two different human inhibitory KIR receptor geneproducts, wherein said antibody is capable of neutralizing KIR-mediatedinhibition of NK cell cytotoxicity on NK cells expressing at least oneof said two different human inhibitory KIR receptors of this invention,including NKVSF1, may be incorporated into liposomes(“immunoliposomes”), alone or together with another substance fortargeted delivery to an animal. Such other substances include nucleicacids for the delivery of genes for gene therapy or for the delivery ofantisense RNA, RNAi or siRNA for suppressing a gene in an NK cell, ortoxins or drugs for the targeted killing of NK cells.

Computer modelling of the extra-cellular domains of KIR2DL1, -2 and -3(KIR2DL1-3), based on their published crystal-structures (Maenaka et al.(1999), Fan et al. (2001), Boyington et al. (2000)), predicted theinvolvement of certain regions or KIR2DL1, -2 and -3 in the interactionbetween KIR2DL1 and the KIR2DL1-3-cross-reactive mouse monoclonalantibodies DF200 and NKVSF1. Thus, in one embodiment, the presentinvention provides antibodies that exclusively bind to KIR2DL1 within aregion defined by the amino acid residues (105, 106, 107, 108, 109, 110,111, 127, 129, 130, 131, 132, 133, 134, 135, 152, 153, 154,155,156, 157,158, 159, 160, 161, 162, 163, 181, 192). In another embodiment theinvention provides antibodies that bind to KIR2DL1 and KIR 2DL2/3without interacting with amino acid residues outside the region definedby the residues (105, 106, 107,108, 109, 110, 111, 127, 129, 130, 131,132, 133, 134, 135, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 181, 192).

In another embodiment, the invention provides antibodies that bind toKIR2DL1 and which does not bind to a mutant of KIR2DL1 in which R131 isAla.

In another embodiment, the invention provides antibodies that bind toKIR2DL1 and which does not bind to a mutant of KIR2DL1 in which R157 isAla.

In another embodiment, the invention provides antibodies that bind toKIR2DL1 and which does not bind to a mutant of KIR2DL1 in which R158 isAla.

In another embodiment, the invention provides antibodies that bind toKIR2DL1 residues (131, 157, 158).

In another embodiment, the invention provides antibodies that bind toKIR2DS3(R131W), but not to wild type KIR2DS3.

In another embodiment, the invention provides antibodies that bind toboth KIR2DL1 and KIR2DL2/3 as well as KIR2DS4.

In another embodiment, the invention provides antibodies that bind toboth KIR2DL1 and KIR2DL2/3, but not to KIR2DS4.

Determination of whether an antibody binds within one of the epitoperegions defined above can be carried out in ways known to the personskilled in the art. As one example of such mapping/characterizationmethods, an epitope region for an anti-KIR antibody may be determined byepitope “foot-printing” using chemical modification of the exposedamines/carboxyls in the KIR2DL1 or KIR2DL2/3 protein. One specificexample of such a foot-printing technique is the use of HXMS(hydrogen-deuterium exchange detected by mass spectrometry) wherein ahydrogen/deuterium exchange of receptor and ligand protein amideprotons, binding, and back exchange occurs, wherein the backbone amidegroups participating in protein binding are protected from back exchangeand therefore will remain deuterated. Relevant regions can be identifiedat this point by peptic proteolysis, fast microbore high-performanceliquid chromatography separation, and/or electrospray ionization massspectrometry. See, e.g., Ehring H, Analytical Biochemistry, Vol. 267 (2)pp. 252-259 (1999) and/or Engen, J. R. and Smith, D. L. (2001) Anal.Chem. 73, 256A-265A. Another example of a suitable epitopeidentification technique is nuclear magnetic resonance epitope mapping(NMR), where typically the position of the signals in two-dimensionalNMR spectres of the free antigen and the antigen complexed with theantigen binding peptide, such as an antibody, are compared. The antigentypically is selectively isotopically labeled with ¹⁵N so that onlysignals corresponding to the antigen and no signals from the antigenbinding peptide are seen in the NMR-spectrum. Antigen signalsoriginating from amino acids involved in the interaction with theantigen binding peptide typically will shift position in the spectres ofthe complex compared to the spectres of the free antigen, and the aminoacids involved in the binding can be identified that way. See, e.g.,Ernst Schering Res Found Workshop. 2004; (44):149-67; Huang et al,Journal of Molecular Biology, Vol. 281 (1) pp. 61-67 (1998); and Saitoand Patterson, Methods. 1996 Jun;9(3):516-24.

Epitope mapping/characterization also can be performed using massspectrometry methods. See, e.g., Downward, J Mass Spectrom. 2000Apr;35(4):493-503 and Kiselar and Downard, Anal Chem. 1999 May1;71(9):1792-801.

Protease digestion techniques also can be useful in the context ofepitope mapping and identification. Antigenic determinant-relevantregions/sequences can be determined by protease digestion, e.g. by usingtrypsin in a ratio of about 1:50 to KIR2DL1 or KIR2DL2/3 o/n digestionat 37° C. and pH 7-8, followed by mass spectrometry (MS) analysis forpeptide identification. The peptides protected from trypsin cleavage bythe anti-KIR binder can subsequently be identified by comparison ofsamples subjected to trypsin digestion and samples incubated withantibody and then subjected to digestion by e.g. trypsin (therebyrevealing a foot print for the binder). Other enzymes like chymotrypsin,pepsin, etc., also or alternatively can be used in a similar epitopecharacterization methods. Moreover, enzymatic digestion can provide aquick method for analyzing whether a potential antigenic determinantsequence is within a region of the KIR2DL1 in the context of a Anti-KIRpolypeptide that is not surface exposed and, accordingly, most likelynot relevant in terms of immunogenicity/antigenicity. See, e.g., Manca,Ann Ist Super Sanita. 1991;27(l):15-9 for a discussion of similartechniques.

Crossreactivity with Cynomolgus Monkeys

It has been found that antibody NKVSF1 also binds to NK cells fromcynomolgus monkeys, see example 7. The invention therefore provides anan antibody, as well as fragments and derivatives thereof, wherein saidantibody, fragment or derivative cross-reacts with at least twoinhibitory human KIR receptors at the surface of human NK cells, andwhich furthermore binds to NK cells from cynomolgus monkeys. In oneembodiment hereof, the antibody is not antibody NKVSF1. The invetionalso provdes a method of testing the toxicity of an antibody, as well asfragments and derivatives thereof, wherein said antibody, fragment orderivative cross-reacts with at least two inhibitory human KIR receptorsat the surface of human NK cells, wherein the method comprises testingthe antibody in a cynomolgus monkey.

Compositions and Administration

The invention also provides pharmaceutical compositions that comprise anantibody, as well as fragments and derivatives thereof, wherein saidantibody, fragment or derivative cross-reacts with at least twoinhibitory KIR receptors at the surface of NK cells, neutralizes theirinhibitory signals and potentiates the activity of those cells, in anysuitable vehicle in an amount effective to detectably potentiate NK cellcytotoxicity in a patient or in a biological sample comprising NK cells.The composition further comprises a pharmaceutically acceptable carrier.Such compositions are also referred to as “antibody compositions of thisinvention.” In one embodiment, antibody compositions of this inventioncomprise an antibody disclosed in the antibody embodiments above. Theantibody NKVSF1 is included within the scope of antibodies that may bepresent in the antibody compositions of this invention.

The term “biological sample” as used herein includes but is not limitedto a biological fluid (for example serum, lymph, blood), cell sample ortissue sample (for example bone marrow).

Pharmaceutically acceptable carriers that may be used in thesecompositions include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

The compositions of this invention may be employed in a method ofpotentiating the activity of NK cells in a patient or a biologicalsample. This method comprises the step of contacting said compositionwith said patient or biological sample. Such method will be useful forboth diagnostic and therapeutic purposes.

For use in conjunction with a biological sample, the antibodycomposition can be administered by simply mixing with or applyingdirectly to the sample, depending upon the nature of the sample (fluidor solid). The biological sample may be contacted directly with theantibody in any suitable device (plate, pouch, flask, etc.). For use inconjunction with a patient, the composition must be formulated foradministration to the patient.

The compositions of the present invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrastemal, intrathecal, intrahepatic,intralesional and intracranial injection or infusion techniques.Preferably, the compositions are administered orally, intraperitoneallyor intravenously.

Sterile injectable forms of the compositions of this invention may beaqueous or an oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a non-toxicparenterally acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or diglycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such ascarboxymethyl cellulose or similar dispersing agents that are commonlyused in the formulation of pharmaceutically acceptable dosage formsincluding emulsions and suspensions. Other commonly used surfactants,such as Tweens, Spans and other emulsifying agents or bioavailabilityenhancers which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms may also be used for thepurposes of formulation.

The compositions of this invention may be orally administered in anyorally acceptable dosage form including, but not limited to, capsules,tablets, aqueous suspensions or solutions. In the case of tablets fororal use, carriers commonly used include lactose and corn starch.Lubricating agents, such as magnesium stearate, are also typicallyadded. For oral administration in a capsule form, useful diluentsinclude lactose and dried cornstarch. When aqueous suspensions arerequired for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, the compositions of this invention may be administered inthe form of suppositories for rectal administration. These can beprepared by mixing the agent with a suitable non-irritating excipientthat is solid at room temperature but liquid at rectal temperature andtherefore will melt in the rectum to release the drug. Such materialsinclude cocoa butter, beeswax and polyethylene glycols.

The compositions of this invention may also be administered topically,especially when the target of treatment includes areas or organs readilyaccessible by topical application, including diseases of the eye, theskin, or the lower intestinal tract. Suitable topical formulations arereadily prepared for each of these areas or organs.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, the compositions may be formulated in asuitable ointment containing the active component suspended or dissolvedin one or more carriers. Carriers for topical administration of thecompounds of this invention include, but are not limited to, mineraloil, liquid petrolatum, white petrolatum, propylene glycol,polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.Alternatively, the compositions can be formulated in a suitable lotionor cream containing the active components suspended or dissolved in oneor more pharmaceutically acceptable carriers. Suitable carriers include,but are not limited to, mineral oil, sorbitan monostearate, polysorbate60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcoholand water.

For ophthalmic use, the compositions may be formulated as micronizedsuspensions in isotonic, pH adjusted sterile saline, or, preferably, assolutions in isotonic, pH adjusted sterile saline, either with orwithout a preservative such as benzylalkonium chloride. Alternatively,for ophthalmic uses, the compositions may be formulated in an ointmentsuch as petrolatum.

The compositions of this invention may also be administered by nasalaerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other conventional solubilizing or dispersingagents.

Several monoclonal antibodies have been shown to be efficient inclinical situations, such as Rituxan (Rituximab), Herceptin(Trastuzumab) or Xolair (Omalizumab), and similar administrationregimens (i.e., formulations and/or doses and/or administrationprotocols) may be used with the antibodies of this invention. Schedulesand dosages for administration of the antibody in the pharmaceuticalcompositions of the present invention can be determined in accordancewith known methods for these products, for example using themanufacturers' instructions. For example, an antibody present in apharmaceutical composition of this invention can be supplied at aconcentration of 10 mg/mL in either 100 mg (10 mL) or 500 mg (50 mL)single-use vials. The product is formulated for IV administration in 9.0mg/mL sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mLpolysorbate 80, and Sterile Water for Injection. The pH is adjusted to6.5. An exemplary suitable dosage range for an antibody in apharmaceutical composition of this invention may between about 10 mg/m²and 500 mg/m². However, it will be appreciated that these schedules areexemplary and that an optimal schedule and regimen can be adapted takinginto account the affinity and tolerability of the particular antibody inthe pharmaceutical composition that must be determined in clinicaltrials. Quantities and schedule of injection of an antibody in apharmaceutical composition of this invention that saturate NK cells for24 hours, 48 hours 72 hours or a week or a month will be determinedconsidering the affinity of the antibody and the its pharmacokineticparameters.

According to another embodiment, the antibody compositions of thisinvention may further comprise another therapeutic agent, includingagents normally utilized for the particular therapeutic purpose forwhich the antibody is being administered. The additional therapeuticagent will normally be present in the composition in amounts typicallyused for that agent in a monotherapy for the particular disease orcondition being treated. Such therapeutic agents include, but are notlimited to, therapeutic agents used in the treatment of cancers,therapeutic agents used to treat infectious disease, therapeutic agentsused in other immunotherapies, cytokines (such as IL-2 or IL-15), otherantibodies and fragments of other antibodies.

For example, a number of therapeutic agents are available for thetreatment of cancers. The antibody compositions and methods of thepresent invention may be combined with any other methods generallyemployed in the treatment of the particular disease, particularly atumor, cancer disease, or other disease or disorder that the patientexhibits. So long as a particular therapeutic approach is not known tobe detrimental to the patient's condition in itself, and does notsignificantly counteract the activity of the antibody in apharmaceutical composition of this invention, its combination with thepresent invention is contemplated.

In connection with solid tumor treatment, the pharmaceuticalcompositions of the present invention may be used in combination withclassical approaches, such as surgery, radiotherapy, chemotherapy, andthe like. The invention therefore provides combined therapies in which apharmaceutical composition of this invention is used simultaneouslywith, before, or after surgery or radiation treatment; or areadministered to patients with, before, or after conventionalchemotherapeutic, radiotherapeutic or anti-angiogenic agents, ortargeted immunotoxins or coaguligands.

When one or more agents are used in combination with anantibody-containing composition of this invention in a therapeuticregimen, there is no requirement for the combined results to be additiveof the effects observed when each treatment is conducted separately.Although at least additive effects are generally desirable, anyincreased anti-cancer effect above one of the single therapies would beof benefit. Also, there is no particular requirement for the combinedtreatment to exhibit synergistic effects, although this is certainlypossible and advantageous.

To practice combined anti-cancer therapy, one would simply administer toan animal an antibody composition of this invention in combination withanother anti-cancer agent in a manner effective to result in theircombined anti-cancer actions within the animal. The agents wouldtherefore be provided in amounts effective and for periods of timeeffective to result in their combined presence within the tumorvasculature and their combined actions in the tumor environment. Toachieve this goal, an antibody composition of this invention andanti-cancer agents may be administered to the animal simultaneously,either in a single combined composition, or as two distinct compositionsusing different administration routes.

Alternatively, the administration of an antibody composition of thisinvention may precede, or follow, the anti-cancer agent treatment by,e.g., intervals ranging from minutes to weeks and months. One wouldensure that the anti-cancer agent and an antibody in the antibodycomposition of this invention exert an advantageously combined effect onthe cancer.

Most anti-cancer agents would be given prior to an inhibitory KIRantibody composition of this invention in an anti-angiogenic therapy.However, when immunoconjugates of an antibody are used in the antibodycomposition of this invention, various anti-cancer agents may besimultaneously or subsequently administered.

In some situations, it may even be desirable to extend the time periodfor treatment significantly, where several days (2, 3, 4, 5, 6 or 7),several weeks (1, 2, 3, 4, 5, 6, 7 or 8) or even several months (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administration of theanti-cancer agent or anti-cancer treatment and the administration of anantibody composition of this invention. This would be advantageous incircumstances where the anti-cancer treatment was intended tosubstantially destroy the tumor, such as surgery or chemotherapy, andadministration of an antibody composition of this invention was intendedto prevent micrometastasis or tumor re-growth.

It also is envisioned that more than one administration of either aninhibitory KIR antibody-based composition of this invention or theanti-cancer agent will be utilized. These agents may be administeredinterchangeably, on alternate days or weeks; or a cycle of treatmentwith an inhibitory KIR antibody composition of this invention, followedby a cycle of anti-cancer agent therapy. In any event, to achieve tumorregression using a combined therapy, all that is required is to deliverboth agents in a combined amount effective to exert an anti-tumoreffect, irrespective of the times for administration.

In terms of surgery, any surgical intervention may be practiced incombination with the present invention. In connection with radiotherapy,any mechanism for inducing DNA damage locally within cancer cells iscontemplated, such as gamma-irradiation, X-rays, UV-irradiation,microwaves and even electronic emissions and the like. The directeddelivery of radioisotopes to cancer cells is also contemplated, and thismay be used in connection with a targeting antibody or other targetingmeans.

In other aspects, immunomodulatory compounds or regimens may beadministered in combination with or as part of the antibody compositionsof the present invention. Preferred examples of immunomodulatorycompounds include cytokines. Various cytokines may be employed in suchcombined approaches. Examples of cytokines useful in the combinationscontemplated by this invention include IL-1 alpha IL-1 beta, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-I11, IL-12, IL-13, IL-15,IL-21, TGF-beta, GM-CSF, M-CSF, G-CSF, TNF-alpha, TNF-beta, LAF, TCGF,BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF, PDGF, IFN-alpha, IFN-beta,IFN-gamma. Cytokines used in the combination treatment or compositionsof this invention are administered according to standard regimens,consistent with clinical indications such as the condition of thepatient and relative toxicity of the cytokine.

In certain embodiments, the cross-reacting inhibitory KIRantibody-comprising therapeutic compositions of the present inventionmay be administered in combination with or may further comprise achemotherapeutic or hormonal therapy agent. A variety of hormonaltherapy and chemotherapeutic agents may be used in the combinedtreatment methods disclosed herein. Chemotherapeutic agents contemplatedas exemplary include, but are not limited to, alkylating agents,antimetabolites, cytotoxic antibiotics, vinca alkaloids, for exampleadriamycin, dactinomycin, mitomycin, carminomycin, daunomycin,doxorubicin, tamoxifen, taxol, taxotere, vincristine, vinblastine,vinorelbine, etoposide (VP-16), 5-fluorouracil (5FU), cytosinearabinoside, cyclophosphamide, thiotepa, methotrexate, camptothecin,actinomycin-D, mitomycin C, cisplatin (CDDP), aminopterin,combretastatin(s) and derivatives and prodrugs thereof.

Hormonal agents include, but are not limited to, for example LHRHagonists such as leuprorelin, goserelin, triptorelin, and buserelin;anti-estrogens such as tamoxifen and toremifene; anti-androgens such asflutamide, nilutamide, cyproterone and bicalutamide; aromataseinhibitors such as anastrozole, exemestane, letrozole and fadrozole; andprogestagens such as medroxy, chlormadinone and megestrol.

As will be understood by those of ordinary skill in the art, theappropriate doses of chemotherapeutic agents will approximate thosealready employed in clinical therapies wherein the chemotherapeutics areadministered alone or in combination with other chemotherapeutics. Byway of example only, agents such as cisplatin, and other DNA alkylatingmay be used. Cisplatin has been widely used to treat cancer, withefficacious doses used in clinical applications of 20 mg/m² for 5 daysevery three weeks for a total of three courses. Cisplatin is notabsorbed orally and must therefore be delivered via injectionintravenously, subcutaneously, intratumorally or intraperitoneally.

Further useful chemotherapeutic agents include compounds that interferewith DNA replication, mitosis and chromosomal segregation, and agentsthat disrupt the synthesis and fidelity of polynucleotide precursors. Anumber of exemplary chemotherapeutic agents for combined therapy arelisted in Table C of U.S. Pat. No. 6,524,583, the disclosure of whichagents and indications are specifically incorporated herein byreference. Each of the agents listed are exemplary and not limiting. Theskilled artisan is directed to “Remington's Pharmaceutical Sciences”15th Edition, chapter 33, in particular pages 624-652. Variation indosage will likely occur depending on the condition being treated. Thephysician administering treatment will be able to determine theappropriate dose for the individual subject.

The present cross-reacting inhibitory KIR antibody compositions of thisinvention may be used in combination with any one or more otheranti-angiogenic therapies or may further comprise anti-angiogenicagents. Examples of such agents include neutralizing antibodies,antisense RNA, siRNA, RNAi, RNA aptamers and ribozymes each directedagainst VEGF or VEGF receptors (U.S. Pat. No. 6,524,583, the disclosureof which is incorporated herein by reference). Variants of VEGF withantagonistic properties may also be employed, as described in WO98/16551, specifically incorporated herein by reference. Furtherexemplary anti-angiogenic agents that are useful in connection withcombined therapy are listed in Table D of U.S. Pat. No. 6,524,583, thedisclosure of which agents and indications are specifically incorporatedherein by reference.

The inhibitory KIR antibody compositions of this invention may also beadvantageously used in combination with methods to induce apoptosis ormay comprise apoptotic agents. For example, a number of oncogenes havebeen identified that inhibit apoptosis, or programmed cell death.Exemplary oncogenes in this category include, but are not limited to,bcr-abl, bcl-2 (distinct from bcl-1, cyclin D1; GenBank accessionnumbers M14745, X06487; U.S. Pat. Nos. 5,650,491; and 5,539,094; eachincorporated herein by reference) and family members including Bcl-x1,Mcl-1, Bak, A1, and A20. Overexpression of bcl-2 was first discovered inT cell lymphomas. The oncogene bcl-2 functions by binding andinactivating Bax, a protein in the apoptotic pathway. Inhibition ofbcl-2 function prevents inactivation of Bax, and allows the apoptoticpathway to proceed. Inhibition of this class of oncogenes, e.g., usingantisense nucleotide sequences, RNAi, siRNA or small molecule chemicalcompounds, is contemplated for use in the present invention to giveenhancement of apoptosis (U.S. Pat. Nos. 5,650,491; 5,539,094; and5,583,034; each incorporated herein by reference).

The inhibitory KIR antibody compositions of this invention may alsocomprise or be used in combination with molecules that comprise atargeting portion, e.g., antibody, ligand, or conjugate thereof,directed to a specific marker of a target cell (“targeting agent”), forexample a target tumor cell. Generally speaking, targeting agents foruse in these additional aspects of the invention will preferablyrecognize accessible tumor antigens that are preferentially, orspecifically, expressed in the tumor site. The targeting agents willgenerally bind to a surface-expressed, surface-accessible orsurface-localized component of a tumor cell. The targeting agents willalso preferably exhibit properties of high affinity; and will not exertsignificant in vivo side effects against life-sustaining normal tissues,such as one or more tissues selected from heart, kidney, brain, liver,bone marrow, colon, breast, prostate, thyroid, gall bladder, lung,adrenals, muscle, nerve fibers, pancreas, skin, or other life-sustainingorgan or tissue in the human body. The term “not exert significant sideeffects,” as used herein, refers to the fact that a targeting agent,when administered in vivo, will produce only negligible or clinicallymanageable side effects, such as those normally encountered duringchemotherapy.

In the treatment of tumors, an antibody composition of this inventionmay additionally comprise or may be used in combination with adjunctcompounds. Adjunct compounds may include by way of example anti-emeticssuch as serotonin antagonists and therapies such as phenothiazines,substituted benzamides, antihistamines, butyrophenones, corticosteroids,benzodiazepines and cannabinoids; bisphosphonates such as zoledronicacid and pamidronic acid; and hematopoietic growth factors such aserythropoietin and G-CSF, for example filgrastim, lenograstim anddarbepoietin.

In another embodiment, two or more antibodies of this invention havingdifferent cross-reactivities, including NKVSF1, may be combined in asingle composition so as to neutralize the inhibitory effects of as manyinhibitory KIR gene products as possible. Compositions comprisingcombinations of cross-reactive inhibitory KIR antibodies of thisinvention, or fragments or derivatives thereof, will allow even widerutility because there likely exists a small percentage of the humanpopulation that may lack each of the inhibitory KIR gene productsrecognized by a single cross-reacting antibody. Similarly, an antibodycomposition of this invention may further comprise one or moreantibodies that recognize single inhibitory KIR subtypes. Suchcombinations would again provide wider utility in a therapeutic setting.

The invention also provides a method of potentiating NK cell activity ina patient in need thereof, comprising the step of administering acomposition according to this invention to said patient. The method ismore specifically directed at increasing NK cell activity in patientshaving a disease in which increased NK cell activity is beneficial,which involves, affects or is caused by cells susceptible to lysis by NKcells, or which is caused or characterized by insufficient NK cellactivity, such as a cancer, another proliferative disorder, aninfectious disease or an immune disorder. More specifically, the methodsof the present invention are utilized for the treatment of a variety ofcancers and other proliferative diseases including, but not limited to,carcinoma, including that of the bladder, breast, colon, kidney, liver,lung, ovary, prostate, pancreas, stomach, cervix, thyroid and skin,including squamous cell carcinoma; hematopoietic tumors of lymphoidlineage, including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkinslymphoma, non-Hodgkins lymphoma, hairy cell lymphoma and Burkettslymphoma; hematopoietic tumors of myeloid lineage, including acute andchronic myelogenous leukemias and promyelocytic leukemia; tumors ofmesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; othertumors, including melanoma, seminoma, teratocarcinoma, neuroblastoma andglioma; tumors of the central and peripheral nervous system, includingastrocytoma, neuroblastoma, glioma, and schwannomas; tumors ofmesenchymal origin, including fibrosarcoma, rhabdomyoscaroma, andosteosarcoma; and other tumors, including melanoma, xerodermapigmentosum, keratoacanthoma, seminoma, thyroid follicular cancer andteratocarcinoma.

Preferred disorders that can be treated according to the inventioninclude hematopoietic tumors of lymphoid lineage, for example T-cell andB-cell tumors, including but not limited to T-cell disorders such asT-prolymphocytic leukemia (T-PLL), including of the small cell andcerebriform cell type; large granular lymphocyte leukemia (LGL)preferably of the T-cell type; Sezary syndrome (SS); Adult T-cellleukemia lymphoma (ATLL); a/d T-NHL hepatosplenic lymphoma;peripheral/post-thymic T cell lymphoma (pleomorphic and immunoblasticsubtypes); angio immunoblastic T-cell lymphoma; angiocentric (nasal)T-cell lymphoma; anaplastic (Ki 1+) large cell lymphoma; intestinalT-cell lymphoma; T-lymphoblastic; and lymphoma/leukaemia (T-Lbly/T-ALL).

Other proliferative disorders can also be treated according to theinvention, including for example hyperplasias, fibrosis (especiallypulmonary, but also other types of fibrosis, such as renal fibrosis),angiogenesis, psoriasis, atherosclerosis and smooth muscle proliferationin the blood vessels, such as stenosis or restenosis followingangioplasty. The cross-reacting inhibitory KIR antibody of thisinvention can be used to treat or prevent infectious diseases, includingpreferably any infections caused by viruses, bacteria, protozoa, moldsor fungi. Such viral infectious organisms include, but are not limitedto, hepatitis type A, hepatitis type B, hepatitis type C, influenza,varicella, adenovirus, herpes simplex type I (HSV-1), herpes simplextype 2 (HSV-2), rinderpest, rhinovirus, echovirus, rotavirus,respiratory syncytial virus, papilloma virus, papilloma virus,cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus,mumps virus, measles virus, rubella virus, polio virus and humanimmunodeficiency virus type I or type 2 (HIV-1, HIV-2).

Bacterial infections that can be treated according to this inventioninclude, but are not limited to, infections caused by the following:Staphylococcus; Streptococcus, including S. pyogenes; Enterococcl;Bacillus, including Bacillus anthracis, and Lactobacillus; Listeria;Corynebacterium diphtheriae; Gardnerella including G. vaginalis;Nocardia; Streptomyces; Thermoactinomyces vulgaris; Treponerna;Camplyobacter, Pseudomonas including Raeruginosa; Legionella; Neisseriaincluding N. gonorrhoeae and N. meningitides; Flavobacterium includingF. meningosepticum and F. odoratum; Brucella; Bordetella including B.pertussis and B. bronchiseptica; Escherichia including E. coli,Klebsiella; Enterobacter, Serratia including S. marcescens and S.liquefaciens; Edwardsiella; Proteus including P. mirabilis and P.vulgaris; Streptobacillus; Rickettsiaceae including R. fickettsfi,Chlamydia including C. psittaci and C. trachomatis; Mycobacteriumincluding M. tuberculosis, M. intracellulare, M. folluitum, M. lapraern,M. avium, M. bovis, M. africanum, M. kansasii, M. intracellulare, and M.lepraemurium; and Nocardia. Protozoa infections that may be treatedaccording to this invention include, but are not limited to, infectionscaused by leishmania, kokzidioa, and trypanosoma. A complete list ofinfectious diseases can be found on the website of the National Centerfor Infectious Disease (NCID) at the Center for Disease Control (CDC)(http://www.cdc.gov/ncidod/diseases/), which list is incorporated hereinby reference. All of said diseases are candidates for treatment usingthe cross-reacting inhibitory KIR antibodies of the invention.

Such methods of treating various infectious diseases may employ theantibody composition of this invention, either alone or in combinationwith other treatments and/or therapeutic agents known for treating suchdiseases, including anti-viral agents, anti-fungal agents, antibacterialagents, antibiotics, anti-parasitic agents and anti-protozoal agents.When these methods involve additional treatments with additionaltherapeutic agents, those agents may be administered together with theantibodies of this invention as either a single dosage form or asseparate, multiple dosage forms. When administered as a separate dosageform, the additional agent may be administered prior to, simultaneouslywith, of following administration of the antibody of this invention.

Further aspects and advantages of this invention will be disclosed inthe following experimental section, which should be regarded asillustrative and not limiting the scope of this application.

EXAMPLE 1 Purification of PBLs and Generation of Polyclonal or Clonal NKCell Lines

PBLs were derived from healthy donors by Ficoll Hypaque gradients anddepletion of plastic adherent cells. To obtain enriched NK cells, PBLswere incubated with anti CD3, anti CD4 and anti HLA-DR mAbs (30 minutesat 4° C.), followed by goat anti mouse magnetic beads (Dynal) (30minutes at 4° C.) and immunomagnetic selection by methods known in theart (Pende et al., 1999). CD3⁻; CD4⁻; DR-cells were cultivated onirradiated feeder cells and 100 U/ml Interleukin 2 (Proleukin, ChironCorporation) and 1.5 ng/ml Phytohemagglutinin A (Gibco BRL) to obtainpolyclonal NK cell populations. NK cells were cloned by limitingdilution and clones of NK cells were characterized by flow cytometry forexpression of cell surface receptors.

The mAbs used were JT3A (IgG2a, anti CD3), EB6 and GL1 83 (IgG1 antiKIR2DL1 and KIR2DL3 respectively), XA-141 IgM (anti KIR2DL1 with thesame specificity as EB6), anti CD4 (HP2.6), and anti DR (D1.12, IgG2a).Instead of JT3A, HP2.6, and DR1.12, which were produced by applicants,commercially available mAbs of the same specificities can be used(Beckman Coulter Inc., Fullerton, Calif.). EB6 and GL183 arecommercially available (Beckman Coulter Inc., Fullerton, Calif. XA-141is not commercially available, but EB6 can be used for controlreconstitution of lysis as described in (Moretta et al., 1993).

Cells were stained with the appropriate antibodies (30 mns at 4° C.)followed by PE or FITC conjugated polyclonal anti mouse antibodies(Southern Biotechnology Associates Inc). Samples were analyzed bycytofluorometric analysis on a FACSAN apparatus (Becton Dickinson,Mountain View, Calif.).

The following clones were used in this study. CP11, CN5 and CN505 areKIR2DL1 positive clones and are stained by EB6 ((IgG1 anti KIR2DL1) orXA-141 (IgM anti KIR2DL1 with same specificity as compared to EB6antibodies). CN12 and CP502 are KIR2DL3 positive clones and are stainedby GL183 antibody (IgG1 anti KIR2DL3).

The cytolytic activity of NK clones was assessed by a standard 4 hour⁵¹Cr release assay in which effector NK cells were tested on Cw3 or Cw4positive cell lines known for their sensitivity to NK cell lysis. Allthe targets were used at 5000 cells per well in microtitration plate andthe effector:target ratio is indicated in the Figures (usually 4effectors per target cells). The cytolytic assay was performed with orwithout supematant of the indicated monoclonal antibodies at a ½dilution. The procedure was essentially the same as described in(Moretta et al., 1993).

EXAMPLE 2 Generation of New mAbs

mAbs were generated by immunizing 5 week old Balb C mice with activatedpolyclonal or monoclonal NK cell lines as described in (Moretta et al.,1990). After different cell fusions, the mAbs were first selected fortheir ability to cross-react with EB6 and GL183 positive NK cell linesand clones. Positive monoclonal antibodies were further screened fortheir ability to reconstitute lysis by EB6 positive or GL183 positive NKclones of Cw4 or Cw3 positive targets respectively.

Cell staining was carried out as follows. Cells were stained with apanel of antibodies (1 μg/ml or 50 μl supernatant, 30 mns at 4° C.)followed by PE-conjugated goat F(ab′)2 fragments anti-mouse IgG (H+L) orPE-conjugated goat F(ab′)2 fragment anti-human IgG (Fc gamma) antibodies(Beckman Coulter). Cytofluorometric analysis was performed on an EpicsXL.MCL apparatus (Beckman Coulter).

One of the monoclonal antibodies, the DF200 mAb, was found to react withvarious members of the KIR family including KIR2DL1, KIR2DL2/3. BothKIR2DL1+ and KIR2DL2/3+ NK cells were stained brightly with DF200 mAb(FIG. 1).

NK clones expressing one or another (or even both) of these HLA classI-specific inhibitory receptors were used as effectors cells againsttarget cells expressing one or more HLA-C alleles. Cytotoxicity assayswere carried out as follows. The cytolytic activity of YTS-KIR2DL1 orYTS-Eco cell lines was assessed by a standard 4 hours 51 Cr releaseassay. The effector cells were tested on HLA-Cw4 positive or negativeEBV cell lines and HLA-Cw4 transfected 721.221 cells. All targets wereused at 3000 cells per well in microtitration plate. The effector/targetratio is indicated in the figures. The cytolytic assay was performedwith or without the indicated full length or F(ab′)2 fragments ofmonoclonal mouse or human antibodies. As expected, KIR2DL1⁺ NK clonesdisplayed little if any cytolytic activity against target cellsexpressing HLA-Cw4 and KIR2DL3⁺ NK clones displayed little or noactivity on Cw3 positive targets. However, in the presence of DF200 mnAb(used to mask their KIR2DL receptors) NK clones became unable torecognize their HLA-C ligands and displayed strong cytolytic activity onCw3 or Cw4 targets.

For example, the C1R cell line (CW4⁺ EBV cell line, ATCC n° CRL 1993)was not killed by KIR2DL1⁺ NK clones (CN5CN505), but the inhibitioncould be efficiently reversed by the use of either DF200 or aconventional anti KIR2DL1 mAb. On the other hand NK clones expressingthe KIR2DL2/3⁺ KIR2DL1- phenotype (CN12) efficiently killed C1R cellsand this killing was unaffected by the DF200 mAb (FIG. 2). Similarresults are obtained with KIR2DL2- or KIR2DL3-positive NK clones on Cw3positive targets.

Similarly, the Cw4+221 EBV cell line was not killed by KIR2DL1⁺transfected NK cells, but the inhibition could be efficiently reversedby the use of either DF200, a DF200 Fab fragment, or a conventional antiKIR2DL1 mAb EB6 or XA141. Also, a Cw3+221 EBV cell line was not killedby KIR2DL2⁺ NK cells, but this inhibition could be reversed by the useof either DF200 or a DF200 Fab fragment. Finally, the latter Cw3+221 EBVcell line was not killed by KIR2DL3⁺ NK cells, but this inhibition couldbe reversed by the use of either a DF200 Fab fragment or a conventionalanti KIR2DL3 mAb GL183 or Y249. The results are shown in FIG. 3.

F(ab′)2 fragments were also tested for their ability to reconstite lysisof Cw4 positive targets. F(ab′)2 fragments of the DF200 and EB6 Abs wereboth able to reverse inhibition of lysis by KIR2DL1-transfected NK cellsof the Cw4 transfected 221 cell line and the Cw4+TUBO EBV cell line.Results are shown in FIG. 4.

EXAMPLE 4 Generation of New Human mAbs

Human monoclonal anti-KIR Abs were generated by immunizing transgenicmice engineered to express a human antibody repertoire with recombinantKIR protein. After different cell fusions, the mAbs were first selectedfor their ability to cross-react with immobilized KIR2DL1 and KIR2DL2protein. Several monoclonal antibodies, including 1-7F9, 1-4F1, 1-6F5and 1-6F1, were found to react with KIR2DL1 and KIR2DL2/3.

Positive monoclonal antibodies were further screened for their abilityto reconstitute lysis by EB6 positive NK transfectants expressingKIR2DL1 of Cw4-positive target cells. The NK cells expressing the HLAclass I-specific inhibitory receptors were used as effectors cellsagainst target cells expressing one or more HLA-C alleles (FIGS. 5 and6). Cytotoxicity assays were carried out as described above. Theeffector/target ratio is indicated in the Figures, and antibodies wereused at either 10 ug/ml or 30 ug/ml.

As expected, KIR2DL1⁺ NK cells displayed little if any cytolyticactivity against target cells expressing HLA-Cw4. However, in thepresence of 1-7F9 mAb, NK cells became unable to recognize their HLA-Cligands and displayed strong cytolytic activity on the Cw4 targets. Forexample, the two cell lines tested (the HLA-Cw4 transfected 721.221 andthe CW4⁺ EBV cell lines) were not killed by KIR2DL1⁺ NK cells, but theinhibition could be efficiently reversed by the use of either Mab 1-7F9or a conventional anti KIR2DL1 mAb EB6. Abs DF200 and panKIR (alsoreferred to as NKVSF1) were compared to 1-7F9. Antibodies 1-4F1, 1-6F5and 1-6F1 on the other hand were not able to reconstitute cell lysis byNK cells on Cw4 positive targets.

EXAMPLE 5 Biacore Analysis of DF200 mAb/KIR2DL1 and DF200 mAb/KIR2DL3Interactions

Production and Purification of Recombinant Proteins

The KIR2DL1 and KIR2DL3 recombinant proteins were produced in E. coli.cDNA encoding the entire extracellular domain of KIR2DL1 and KIR2DL3were amplified by PCR from pCDM8 clone 47.11 vector (Biassoni et al,1993) and RSVS(gpt)183 clone 6 vector (Wagtman et al, 1995)respectively, using the following primers: Sense:5′-GGAATTCCAGGAGGAATTTAAAATGCATGAGGGAGTCCACAG-3′ Anti-sense:5′-CGGGATCCCAGGTGTCTGGGGTTACC-3′They were cloned into the pML1 expression vector in frame with asequence encoding a biotinylation signal (Saulquin et al, 2003).

Protein expression was performed in the BL21(DE3) bacterial strain(Invitrogen). Transfected bacteria were grown to OD₆₀₀=0.6 at 37° C. inmedium supplemented with ampicillin (100 μg/ml) and expression wasinduced with 1 mM IPTG.

Proteins were recovered from inclusion bodies under denaturingconditions (8 M urea). Refolding of the recombinant proteins wasperformed in 20 mM Tris, pH 7.8, NaCl 150 mM buffer containingL-arginine (400 mM, Sigma) and β-mercaptoethanol (1 mM), at roomtemperature, by decreasing the urea concentration in a six step dialysis(4, 3, 2, 1 0.5 and 0 M urea, respectively). Reduced and oxidizedglutathione (5 mM and 0.5 mM respectively, Sigma) were added during the0.5 and 0 M urea dialysis steps. Finally, the proteins were dialyzedextensively against 10 mM Tris, pH 7.5, NaCl 150 mM buffer. Soluble,refolded proteins were concentrated and then purified on a Superdex 200size-exclusion column (Pharmacia; AKTA system).

Surface plasmon resonance measurements were performed on a Biacoreapparatus (Biacore). In all Biacore experiments HBS buffer supplementedwith 0.05% surfactant P20 served as running buffer.

Protein immobilisation.

Recombinant KIR2DL1 and KIR2DL3 proteins produced as described abovewere immobilized covalently to carboxyl groups in the dextran layer on aSensor Chip CM5 (Biacore). The sensor chip surface was activated withEDC/NHS (N-ethyl-N′-(3-dimethylaminopropyl)carbodiimidehydrochloride andN-hydroxysuccinimide, Biacore). Proteins, in coupling buffer (10 mMacetate, pH 4.5) were injected. Deactivation of the remaining activatedgroups was performed using 100 mM ethanolamine pH 8 (Biacore).

Affinity Measurements.

For kinetic measurements, various concentrations of the soluble antibody(1×10⁻⁷ to 4×10⁻¹⁰ M) were applied onto the inmobilized sample.Measurements were performed at a 20 μl/min continuous flow rate. Foreach cycle, the surface of the sensor chip was regenerated by 5 μlinjection of 10 mM NaOH pH 11. The BIAlogue Kinetics Evaluation program(BIAevaluation 3.1, Biacore) was used for data analysis. The solubleanalyte (40 μl at various concentrations) was injected at a flow rate of20 μl/min in HBS buffer, on dextran layers containing 500 or 540reflectance units (RU), and 1000 or 700 RU of KIR2DL1 and KIR2DL3,respectively. Data are representative of 6 independent experiments. Theresults are shown in Table 1, below. TABLE 1 BIAcore analysis of DF200mAb binding to immobilized KIR2DL1 and KIR2DL3. Protein K_(D) (10⁻⁹ M)KIR2DL1 10.9 +/− 3.8 KIR2DL3  2.0 +/− 1.9K_(D): Dissociation constant.

EXAMPLE 6 Biacore Competitive Binding Analysis of Murine and Humananti-KIR Antibodies

Epitope mapping analysis was performed on immobilized KIR 2DL1 (900 RU),KIR 2DL3 (2000 RU) and KIR 2DS1 (1000 RU) with mouse anti-KIR 2Dantibodies DF200, Pan2D, gl 183 and EB6, and human anti-KIR 2Dantibodies 1-4F1, 1-6F1, 1-6F5 and 1-7F9 as described previously(Gauthier et al 1999, Saunal and van Regenmortel 1995).

All experiments were done at a flow rate of 5 μl/min in HBS buffer with2 min injection of the different antibodies at 15 μg/ml. For each coupleof antibodies competitive binding analysis was performed in two steps.In the first step the first monoclonal antibody (mAb) was injected onKIR 2D target protein followed by the second mAb (without removing thefirst mAb) and second mAb RU value (RU2) was monitored. In the secondstep the second mAb was injected first, directly on nude KIR 2D protein,and mAb RU value (RU1) was monitored. Percent inhibition of second mAbbinding to KIR 2D protein by first mab was calculated by:100*(1-RU2/RU1).

Results are shown in Tables 2,3 and 4, where the antibodies designated‘first antibody’ are listed on vertical column and the ‘second antibody’are listed on the horizontal column. For each antibody combinationtested, the values for direct binding level (RU) of the antibodies tothe chip are listed in the table, where direct binding of the secondantibody to the KIR2D chip is listed in the upper portion of the fieldand the value for binding of the second antibody to the KIR2D chip whenthe first antibody is present is listed in the lower portion of thefield. Listed in the right of each field is the percentage inhibition ofsecond antibody binding. Table 2 shows binding on a KIR2DL1 chip, Table3 shows binding of antibodies to a KIR2DL3 chip, and Table 4 showsbinding of antibodies to a KIR2DS1 chip.

Competitive binding of murine antibodies DF200, NKVSF1 and EB6, andhuman antibodies 1-4F1, 1-7F9 and 1-6F1 to immobilized KIR2DL1,KIR2DL2/3 and KIR2DS1 was assessed. Epitope mapping (FIG. 7) fromexperiments with anti-KIR antibodies' binding to KIR2DL1 showed that (a)antibody 1-7F9 is competitive with EB6 and 1-4F1, but not with NKVSF1and DF200; (b) antibody 1-4 F1 in turn is competitive with EB6, DF200,NKVSF1 and 1-7 F9; (c) NKVSF1 competes with DF200,1-4F1 and EB6, but not1-7F9; and (d) DF200 competes with NKVSF1, 1-4F1 and EB6, but not 1-7F9.Epitope mapping (FIG. 8) from experiments with anti-KIR antibodies'binding to KIR2DL3 showed that (a) 1-4F 1 is competitive with NKVSF1,DF200, gl183 and 1-7F9; (b) 1-7F9 is competitive with DF200, gl183 and1-4F 1, but not with NKVSF1; (c) NKVSF1 competes with DF200, 1-4F1 andGL183, but not 1-7F9; and (d) DF200 competes with NKVSF1,1-4F1 and1-7F9, but not with GL183. Epitope mapping (FIG. 9) from experimentswith anti-KIR antibodies' binding to KIR2DS I showed that (a) 1-4F1 iscompetitive with NKVSF1, DF200 and 1-7F9; (b) 1-7F9 is competitive with1-4F1 but not competitive with DF200 and NKVSF1; (c) NKVSF1 competeswith DF200 and 1-4F1, but not 1-7F9; and (d) DF200 competes with NKVSF1and 1-4F1, but not with 1-7F9.

EXAMPLE 7 Anti-KIR mAb titration with cvnomolgus NK cells

Anti-KIR antibody NKVSF1 was tested for its ability to bind to NK cellsfrom cynomolgus monkeys. Binding of the antibody to monkey NK cells isshown in FIG. 10.

Purification of monkey PBMC and generation of polyclonal NK cell bulk.

Cynomolgus Macaque PBMC were prepared from Sodium citrate CPT tube(Becton Dickinson). NK cells purification was performed by negativedepletion (Macaque NK cell enrichment kit, Stem Cell Technology). NKcells were cultivated on irradiated human feeder cells, 300 U/mlInterleukin 2 (Proleukin, Chiron Corporation) and 1ng/mlPhytohemagglutinin A (Invitrogen, Gibco) to obtain polyclonal NK cellpopulations.

Pan2D mAb titration with cynomolgus NK cells.

Cynomolgus NK cells (NK bulk day 16) were incubated with differentamount of Pan2D mAb followed by PE-conjugated goat F(ab′)2 fragmentsanti-mouse IgG (H+L) antibodies. The percentage of positive cells wasdetermined with an isotypic control (purified mouse IgG1).Samples weredone in duplicate. Mean fluorescence intensity =MFI. TABLE 2 KIR2DL1epitope mapping First Ab

Second Ab → (below) DF200 Pan2D EB6 1-4 F1 1-7 F9 1-6 F1 1-6 F5 DF20080%  90% 490 92% 480 27% 540 15% 400 15% 40 350 460 340 Pan2D 90% 90%900 95% 860  2% 750 12% 600 13% 50 840 660 520 EB6 60% 40%  460 57% 37048% 490 65% 260 23% nd 200 190 170 200 1-4 F1 1-7 F9 600 10% 545 2% 46060% 360 95% 330  9% nd 545 534 180 16 300 1-6 F1 350 11% 475 7% 260 18%360 23% 490 10% nd 310 440 320 275 440 1-6 F5 350 17% 475 7% nd 360 17%nd 290 40% 290 440 300 170

TABLE 3 KIR2DL3 epitope mapping First Ab

Second Ab → (below) DF200 Pan2D gl183 1-4 F1 1-7 F9 1-6 F1 1-6 F5 DF20075% 20% 1270 75% 520 62% 550 16% 440 4% 320 200 460 420 Pan2D 95% 85%2250 68% 880 15% 840  8% 560 18%  730 750 770 460 gl183  8% 40% 1300 75%670 76% 530 18% nd 330 160 430 1-4 F1 1140 82% 2400 63% 1240 73% 105087% 210 890 330 140 1-7 F9 770 42% 870  5% 800 75% 1000 63% 450 830 200270 1-6 F1 790  4% 990  0% 620  8% 760 1090 570 1-6 F5 800  5% 990  4%nd 760 950

TABLE 4 KIR2DS1 epitope mapping First Ab

Second Ab → (below) DF200 Pan2D 1-4 F1 1-7 F9 DF200 70% 660 87% 975 15% 80 825 Pan2D 100% 650 100%  920 45% * −8 500 1-7 F9 900  17% 1350 11%660 96% 1090 1200 23

EXAMPLE 8 Epitope-mapping of DF200- and pan2D-binding to KIR2DL1

Computer modelling of the extra-cellular domains of KIR2DL1, -2 and -3(KIR2DL1-3), based on their published crystal-structures (Maenaka et al.(1999), Fan et al. (2001), Boyington et al. (2000)), predicted theinvolvement of amino acids R131¹ in the interaction between KIR2DL1 andthe KIR2DL1-3-cross-reactive mouse monoclonal antibodies (mAb's) DF200and pan2D. To verify this, fusion-proteins were prepared consisting ofthe complete extra-cellular domain of KIR2DL1 (amino acids H1-H224),either wild-type or point-mutated (e.g. R131W²), fused to human Fc(hFc). The material and methods used to produce and evaluate the variousKIR2DL1-hFc fusion-proteins have been described (Winter and Long(2000)). In short, KIR2DL1(R131W)-hFc encoding cDNA-vectors weregenerated, by PCR-based mutagenesis (Quickchange II, Promega) ofCL42-Ig, a published cDNA-vector for the production of wild-typeKIR2DLI-hFc (Wagtmann et al. (1995)). KIR2DL1-hFc and KIR2DL1(R131W)-hFcwere produced in COS7 cells and isolated from tissue-culture media,essentially as described (Wagtmann et al. (1995)). To test their correctfolding, KIR2DL1-hFc and KIR2DL1(R131W)-hFc were incubated withLCL721.221 cells that express either HLA-Cw3 (no KIR2DL1 ligand) orHLA-Cw4 (KIR2DL1 ligand), and the interaction between KIR-Fc fusionproteins and cells analysed by FACS, a standard technique for the studyof protein-interactions at the cell-surface. An example of independentexperiments is given in FIG. 11, panel A. As predicted from theliterature, none of the KIR2DL1-hFc fusion proteins bound HLA-Cw3expressing LCL721.221 cells. In contrast, both KIR2DL1-hFc andKIR2DL1(R131W)-hFc bound to HLA-Cw4 expressing LCL721.221 cells, therebyconfirming their correct folding. The binding of KIR2DL1(RI31W)-hFc andKIR2DL1-hFc to KIR-specific mAb's (DF200, pan2D, EB6 and GL183) wasstudied using ELISA, a standard technique to study protein-interactions.In short, KIR2DL1(R131W)-hFc and KIR2DL1-hFc were linked to 96-wellsplates via goat anti-human antibodies, after which KIR-specific mAb'swere added in various concentrations (0-1 μg/ml in PBS). Theinteractions between KIR2DL1-hFc variants and mAb's were visualised byspectrophotometry (450 nm), using peroxidase-coupled secondaryantibodies specific for mouse antibodies to convert TMB substrate. Anexamples of independent experiments is given in FIG. 11, panel B.Whereas the KIR2DL2-3-specific mAb GL83 was not able to bind any of theKIR2DL1-hFc fusion proteins, the KIR2DL1-specific mAb EB6, DF200 andpan2D bound KIR2DL1-hFc variants in a dose-dependent fashion. The singlepoint-mutation (R131W) affected the binding of DF200 and pan2D with areduction in binding compared to wild type of ˜10% at highestconcentrations of mab (1 μg/ml), confirming that R131 is part of thebinding-site of DF200 and pan2D in extra-cellular domain 2 of KIR2DL1.

REFERENCES

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Moretta, A., Vitale, M., Bottino, C., Orengo, A. M., Morelli, L.,Augugliaro, R., Barbaresi, M., Ciccone, E., and Moretta, L. (1993). P58molecules as putative receptors for major histocompatibility complex(MHC) class I molecules in human natural killer (NK) cells. Anti-p58antibodies reconstitute lysis of MHC class I-protected cells in NKclones displaying different specificities. J Exp Med 178, 597-604.

Pende, D., Parolini, S., Pessino, A., Sivori, S., Augugliaro, R.,Morelli, L., Marcenaro, E., Accame, L., Malaspina, A., Biassoni, R., etal (1999). Identification and molecular characterization of NKp30, anovel triggering receptor involved in natural cytotoxicity mediated byhuman natural killer cells. J Exp Med 190, 1505-1516.

Ruggeri, L., Capanni, M., Urbani, E., Perruccio, K., Shlomchik, W. D.,Tosti, A., Posati, S., Rogaia, D., Frassoni, F., Aversa, F., et al.(2002). Effectiveness of donor natural killer cell alloreactivity inmismatched hematopoietic transplants. Science 295, 2097-2100.

Wagtmann N, Biassoni R, Cantoni C, Verdiani S, Malnati MS, Vitale M,Bottino C, Moretta L, Moretta A, Long EO.Molecular clones of the p58 NKcell receptor reveal immunoglobulin-related molecules with diversity inboth the extra- and intracellular domains.Immunity. 1995May;2(5):439-49.

Biassoni R, Verdiani S, Cambiaggi A, Romeo PH, Ferrini S, Moretta L.Human CD3-CD16+natural killer cells express the hGATA-3 T celltranscription factor and an unrearranged 2.3-kb TcR delta transcript.Eur J Immunol. 1993 May;23(5):1083-7. Saulquin X, Gastinel L N, VivierE.Crystal structure of the human natural killer cell activating receptorKIR2DS2 (CD158j) J Exp Med. 2003 Apr 7;197(7):933-8.

Gauthier, L., Lemmers, B., Guelpa-Fonlupt, V., Fougereau, M., andSchiff, C. μ-SLC physico-chemical interactions of the human preB cellreceptor: implications for VH repertoire selection and cell signaling atthe preB cell stage. Journal of Immunology, 162., 41-50. (1999).

Saunal, H. and Van Regenmortel, M. H. V., Mapping of viral conformationepitopes using biosensor measurements. Journal of Immunology, 183: 33-41(1995).

Boyington J C; Motyka S A; Schuck P; Brooks A G; Sun P D. Nature, Vol.405 (6786) pp. 537-543 (2000)

Fan Q R; Long E O; Wiley D C. Nature immunology, Vol. 2 (5) pp. 452-460(2001) Maenaka K; Juji T; Stuart D I; Jones E Y. Structure with Foldingand design, Vol. 7 (4) pp. 391-398 (1999)

Wagtmann N; Rajagopalan S; Winter C C; Peruzzi M; Long E O. Immunity,Vol. 3 (6) pp. 801-809 (1995)

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All references, including publications, patent applications and patents,cited herein are hereby incorporated by reference to the same extent asif each reference were individually and specifically indicated to beincorporated by reference and were set forth in its entirety herein.

All headings and sub-headings are used herein for convenience only andshould not be construed as limiting the invention in any way,

Any combination of the above-described elements in all possiblevariations thereof is encompassed by the invention unless otherwiseindicated herein or otherwise clearly contradicted by context.

The terms “a” and “an” and “the” and similar referents as used in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. Unless otherwise stated, all exact valuesprovided herein are representative of corresponding approximate values(e.g., all exact exemplary values provided with respect to a particularfactor or measurement can be considered to also provide a correspondingapproximate measurement, modified by “about,” where appropriate).

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise indicated. No language in the specification should beconstrued as indicating any element is essential to the practice of theinvention unless as much is explicitly stated.

The citation and incorporation of patent documents herein is done forconvenience only and does not reflect any view of the validity,patentability and/or enforceability of such patent documents,

The description herein of any aspect or embodiment of the inventionusing terms such as “comprising”, “having”, “including” or “containing”with reference to an element or elements is intended to provide supportfor a similar aspect or embodiment of the invention that “consists of”,“consists essentially of”, or “substantially comprises” that particularelement or elements, unless otherwise stated or clearly contradicted bycontext (e.g., a composition described herein as comprising a particularelement should be understood as also describing a composition consistingof that element, unless otherwise stated or clearly contradicted bycontext).

This invention includes all modifications and equivalents of the subjectmatter recited in the aspects or claims presented herein to the maximumextent permitted by applicable law.

1. A method of producing an antibody which cross-reacts with multipleKIR2DL gene products (KIRS) and which neutralizes the inhibitoryactivity of such KIRs, said method comprising the steps of: (a)immunizing a non-human mammal with an immunogen comprising a KIR2DLpolypeptide; (b) preparing antibodies from said immunized mammal,wherein said antibodies bind said KIR2DL polypeptide, (c) selectingantibodies of (b) that cross-react with at least two different KIR2DLgene products, (d) selecting antibodies of (c) that potentiate NK cells,and (e) selecting and isolating an antibody from the antibodies of (d)that binds a primate NK cell or KIR polypeptide.
 2. The method of claim1, wherein the primate in step (e) is a cynomolgus monkey.
 3. Anisolated antibody, antibody fragment, or an antibody derivativecomprising one or more light variable region CDRs of Pan2D as set forthin FIG.
 12. 4. The antibody, antibody fragment, or an antibodyderivative of claim 3, wherein the antibody, antibody fragment, orantibody derivative comprises the light chain variable region sequenceof Pan2D as set forth in FIG.
 12. 5. An isolated antibody, antibodyfragment, or an antibody derivative comprising one or more of the lightvariable region CDRs of DF-200 as set forth in FIG.
 12. 6. The antibody,antibody fragment, or antibody derivative of claim 5, wherein theantibody, antibody fragment, or antibody derivative comprises the lightchain variable region sequence of DF-200 as set forth in FIG.
 12. 7. Anisolated antibody, antibody fragment, or an antibody derivativecomprising one or more of the heavy variable region CDRs of DF-200 asset forth in FIG.
 13. 8. The antibody, antibody fragment, or an antibodyderivative of claim 10, wherein the antibody, antibody fragment, orantibody derivative comprises the heavy chain variable region sequenceof DF-200 as set forth in FIG.
 13. 9. An isolated antibody, antibodyfragment, or antibody derivative that exclusively binds to KIR2DL1within a region defined by amino acid residues 105, 106, 107, 108, 109,110, 111, 127, 129, 130, 131, 132, 133, 134, 135, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 181,
 192. 10. An isolatedantibody, antibody fragment, or antibody derivative that binds toKIR2DL1 and KIR 2DL2/3 without interacting with amino acid residuesoutside the region defined by residues 105, 106, 107, 108, 109, 110,111, 127, 129, 130, 131, 132, 133, 134, 135, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 181,
 192. 11. An isolated antibody,antibody fragment, or antibody derivative that binds to KIR2DL1 and doesnot bind to a mutant of KIR2DL1 characterized by one or more of R131 isAla, R157 is Ala, and R158 is Ala.
 12. An isolated antibody, antibodyfragment, or antibody derivative that binds to KIR2DL1 residues 131,157,
 158. 13. An isolated antibody, antibody fragment, or antibodyderivative that binds to KIR2DS3(R131W), but that does not bind to wildtype KIR2DS3.
 14. An isolated antibody, antibody fragment, or antibodyderivative that binds KIR2DL1, KIR2DL2/3, and KIR2DS4.
 15. An isolatedantibody, antibody fragment, or antibody derivative that binds to bothKIR2DL1 and KIR2DL2/3, but not to KIR2DS4.
 16. A pharmaceuticallyacceptable composition comprising an effective amount of an antibody,antibody fragment, or antibody derivative according to claim
 15. 17. Apharmaceutically acceptable composition comprising an effective amountof an antibody, antibody fragment, or antibody derivative according toclaim
 14. 18. A pharmaceutically acceptable composition comprising aneffective amount of an antibody, antibody fragment, or antibodyderivative according to claim
 3. 19. A pharmaceutically acceptablecomposition comprising an effective amount of an antibody, antibodyfragment, or antibody derivative according to claim
 4. 20. Apharmaceutically acceptable composition comprising an effective amountof an antibody, antibody fragment, or antibody derivative according toclaim
 5. 21. A pharmaceutically acceptable composition comprising aneffective amount of an antibody, antibody fragment, or antibodyderivative according to claim
 6. 22. A pharmaceutically acceptablecomposition comprising an effective amount of an antibody, antibodyfragment, or antibody derivative according to claim
 7. 23. Apharmaceutically acceptable composition comprising an effective amountof an antibody, antibody fragment, or antibody derivative according toclaim 8.